Embryonic Growth-Associated Protein Is One Subunit of a Novel N-Terminal Acetyltransferase Complex Essential for Embryonic Vascula
http://www.100md.com
Janet M. Wenzlau, Pamela J. Garl, Peter
参见附件。
the Departments of Renal Medicine (R.A.N., M.C.M.W.-E.), Cell and Developmental Biology (K.B.A., M.C.M.W.-E.), Craniofacial Biology (P.S., K.B.A.), Pediatrics (J.M.W., P.J.G., K.R.S., M.C.M.W.-E.)
Cardiovascular Pulmonary Research (J.W.), University of Colorado Health Sciences Center, Denver.
Abstract
N-terminal protein acetylation, catalyzed by N-terminal acetyltransferases (NATs) recognizing distinct N-terminal sequences, is gaining recognition as an essential regulator of normal cell function, but little is known of its role in vertebrate development. We previously cloned a novel gene, embryonic growth-associated protein (EGAP), the expression of which is associated with rapid vascular smooth muscle cell proliferation during development. We show herein EGAP is the mammalian/zebrafish homologue of yeast Mak10p, one subunit of the yeast NatC complex, and describe the cloning of its binding partners Mak3 and Mak31. The EGAP NAT forms a functional complex in mammalian cells, is evolutionarily conserved, and developmentally regulated. It is widely but not ubiquitously expressed during early zebrafish development but undetectable in later developmental stages. We demonstrate EGAP- and Mak3-deficient zebrafish fail to develop because of, in part, decreased cell proliferation, increased apoptosis, and poor blood vessel formation contributing to embryonic lethality. We examined the role of target of rapamycin (TOR), a highly conserved protein kinase controlling cell growth, as a physiological target of EGAP NAT acetylation. Compared with controls, TOR expression and signaling is significantly reduced in EGAP morphants. Pharmacological inhibition of TOR with rapamycin phenocopied the EGAP morpholino oligonucleotide-induced growth and vessel defects. Overexpression of constitutively active TOR rescued EGAP morphants, suggesting TOR is a direct or indirect endogenous substrate of the EGAP NAT complex. These data suggest the EGAP NAT complex is an essential regulatory enzyme controlling the function of a subset of proteins required for embryonic growth control and vessel development.
Key Words: EGAP TOR N-terminal acetyltransferase vascular development vascular cell proliferation
Introduction
Smooth muscle cell (SMC) proliferation plays a prominent role in normal vessel development and in vascular pathologies, including atherosclerosis and restenosis after angioplasty and stent placement.1–3 During vessel development, SMCs undergo a transient phase of rapid proliferation during which the vessel wall acquires its complement of SMCs.4–8 Replication decreases with vessel maturation as SMCs reach a highly quiescent, differentiated state in adult vessels. However, a hallmark of vascular occlusive disease and interventions in response to disease is a transient increase in SMC replication to levels similar to those exhibited during embryonic life.9,10 SMCs cultured from embryonic vessels or from the neointima of injured vessels during periods of rapid in vivo growth exhibit a unique growth phenotype characterized by rapid serum-stimulated growth and the ability to replicate in a mitogen-independent manner.4,10 This is distinct from SMCs derived from adult uninjured vessels, which exhibit slower serum-stimulated growth and become quiescent under mitogen-deprived conditions. However, the mechanisms regulating this unique SMC growth phenotype at these times have yet to be fully elucidated.
To facilitate our characterization of this rapid proliferative phenotype, we previously cloned several novel genes selectively expressed by rapidly proliferating cells during early vascular development and only re-expressed in the adult vasculature by cells responding to pathological injury.11 One of these genes, which we named embryonic growth-associated protein (EGAP), is expressed in a variety of tissues in the developing rat embryo but restricted in expression in the adult, remaining detectable only in tissues undergoing continual cell renewal or in cells responding to pathological injury. We report EGAP, the vertebrate orthologue of the yeast Mak10p protein, is one subunit of a novel N-terminal acetyltransferase (NAT) that is highly conserved among vertebrate species.
N-terminal acetylation is a post-translational modification of proteins in which the acetyl group of AcetylCoA is transferred to the initiator methionine or most N-terminal amino acid.12–14 This irreversible protein modification is catalyzed by a family of NATs, each recognizing distinct N-termini sequences of specific protein substrates. Most information regarding N-acetylation has been obtained from studies in yeast, which express at least three NATs: NatA, NatB, and NatC.12,14,15 Each complex consists of independently transcribed subunits; loss or mutation of any one subunit results in loss of NAT activity. N-acetylation may alter protein functions such as protein stability, protein-protein interactions, further modifications such as phosphorylation, and translocation to specific cellular compartments.12 For instance, yeast NatB acetylation of actin and tropomyosin is essential for interactions of these proteins and actin filament stabilization.16,17 Although many proteins are N-terminal acetylated, there is only a small body of knowledge regarding the role of this protein modification in vertebrate systems. Existing information focuses on the role of the vertebrate Ard1–NatH complex, the yeast NatA-related complex that has the largest number of endogenous substrates identified to date.18–21 In contrast, much less is known of the NatC complex, consisting of Mak3p, Mak10p, and Mak31p, or its endogenous substrates.22 Subunit copurification and similar slow growth phenotypes of all three yeast deletion strains (mak3p-, mak10p-, and mak31p-) highly suggest that interaction of subunits is required for NatC enzyme activity. However, virtually nothing is known of the biological significance of the EGAP/NatC complex in vertebrate species. Our data establish the EGAP NAT complex functionally coassembles in mammalian cells to regulate cell proliferation and is essential for embryonic development, at least in part through the regulation of target of rapamycin (TOR) signaling events.
Materials and Methods
An expanded Materials and Methods section can be found in the online supplement, available at http://circres.ahajournals.org.
Genomic Sequence Analysis and Morpholino Design
A positive mouse bacterial artificial chromosome (BAC) clone corresponding to rat EGAP cDNA sequence was obtained from Research Genetics (mouse RPCI BAC 465-E7) and mapped to deduce the genomic structure and complete cDNA sequence. Full-length Flag-, Myc-, and influenza hemagglutinin (HA)-tagged expression constructs were generated for mouse Mak31, EGAP, and Mak3, respectively, by RT-PCR using embryonic day 14 mouse poly(A+) RNA. The genomic organization of the zebrafish EGAP and Mak3 loci were deduced from comparisons of their cDNA sequences with genomic trace sequences using the BLAST program on the NCBI website. Antisense morpholino oligonucleotides (MOs) were designed against the splice donor at the end of the first coding exons.
In Situ Hybridization, Immunohistochemistry, and TUNEL Staining
Whole-mount in situ hybridization was performed as described previously. Whole-mount immunohistochemistry was performed using the Vectastain Avidin/Biotin/Horseradish peroxidase ABC Elite System as described previously. TUNEL was used to assess apoptosis in control and EGAP morphants. Briefly, embryos were permeabilized, washed, incubated in terminal transferase (Roche), and biotin-labeled ddUTP (Roche) and biotin incorporation detected with the peroxidase ABC kit (Vector) using diaminobenzidine as chromogen. For 5-bromodeoxyuridine (BrdUrd) immunohistochemistry, embryos were incubated with BrdUrd, and 5-μm cryosections were immunohistochemically stained for BrdUrd as described previously.4
Results
Identification and Functional Role of the EGAP NAT Complex
Verification that EGAP mRNA is developmentally regulated in rat aortic SMCs and sequence analysis showing the EGAP NAT complex is highly conserved can be found in supplemental Figures 1 and 2, available online at http://circres.ahajournals.org.
Studies from yeast proposed NatC subunit expression and assembly are required for its acetyltransferase activity.22 Thus, we used coexpression and coimmunoprecipitation analyses to determine whether the mammalian complex similarly colocalizes and functionally assembles in vivo. Human embryonic kidney 293 (HEK 293) cells were transiently transfected with Myc-tagged mouse EGAP (mEGAP), HA-tagged mMak3, and Flag-tagged mMak31 expression constructs to determine cellular localization and to determine overlap in cellular distribution (Figure 1A). All proteins appear in the cytoplasm, are excluded from the nucleus, and show a high degree of overlap in cellular distribution (Figure 1A, merge). To determine whether the mammalian proteins coassemble in living cells, whole cell lysates from transfected HEK 293 cells were immunoprecipitated using anti-Myc or anti-Flag antibodies, and Western blotting was performed using anti-Myc, anti-Flag, or anti-HA antibodies (Figure 1B). Myc-mEGAP, HA-mMak3, and Flag-mMak31 were detected in cells overexpressing all three subunits using either anti-Myc or anti-Flag for immunoprecipitation (Figure 1B, lanes 1 and 8), demonstrating mEGAP, mMak3, and mMak31 coassemble in mammalian cells. The results from these reciprocal immunoprecipitation experiments strongly suggest formation of a stable in vivo complex in mammalian cells. Omission of mMak31 (Figure 1B, lanes 3 and 9), mMak3 (Figure 1B, lanes 4 and 10), or mEGAP (Figure 1B, lanes 5 and 11) from transfections resulted in loss of subunit assembly, especially recruitment and association of mMak3, the catalytic subunit, with mEGAP and mMak31 (mEGAP and mMak31 continued to associate in the absence of mMak3; Figure 1B, lanes 4 and 10).
Because the mammalian EGAP NAT proteins form a stable in vivo complex and EGAP is associated with rapid cell proliferation,11 we determined the functional role of subunit assembly on cell growth. Transfected HEK 293 cells were incubated in low serum media in the presence of BrdUrd for 24 hours, and the degree of BrdUrd incorporation, and therefore DNA synthesis, was determined. Compared with nontransfected cells that begin to exhibit a decline in replication after 24 hours in low serum, cells overexpressing all three subunits maintained high rates of BrdUrd incorporation (Figure 1C). Omission of mEGAP, mMak3, or mMak31 from transfections resulted in reduced levels of BrdUrd incorporation (similar to nontransfected cells), highly suggestive of a functional role for the EGAP NAT complex in regulating cell proliferation.
The EGAP NAT Complex Is Essential for Zebrafish Development
Our previous data11 suggested expression and subsequent downregulation of EGAP plays an important role in development and differentiation, including vascular cell growth and differentiation. We next used the zebrafish model to determine the biological significance of the EGAP NAT during development. Using whole-mount in situ hybridization, we found zEGAP mRNA is expressed at high levels in the developing zebrafish embryo as early as 6 hours post fertilization (hpf) and remains high in many developing tissues through 18 hpf and 28 hpf (Figure 2Aa through 2Ac). Consistent with our previous studies in the rat, zEGAP expression was high in developing vascular structures, implicating an important role in blood vessel development (Figure 2Ad through 2Ag). Staining was also strong in the epidermis but not the neural tube or notochord (Figure 2Ag). Staining becomes restricted to the developing head by 48 hpf (Figure 2Ah) and by 5 days post fertilization (dpf), is observed only in the epidermis (Figure 2Ai), where actively replicating cells were detected (Figure 2Aj). The temporal and spatial pattern of zMak3 expression is very similar to that of zEGAP (Figure 2B), and both are similar to those we reported previously for EGAP in the developing rat; thus, EGAP and Mak3 are evolutionarily conserved, developmentally regulated genes.
To establish the EGAP NAT is essential for zebrafish development, we diminished EGAP expression using MOs targeted to the splice donor site at the end of EGAP exon 2 (containing the ATG start codon). Microinjection of 6 ng MO into zebrafish embryos produced a truncated form of EGAP through insertion of a partial intron sequence (Figure 3A). Compared with control MO-injected embryos, EGAP morphants exhibited significant delays in growth (Figure 3B and 3C), and very few live EGAP morphants were observed after 4 to 5 dpf. At the 14-somite stage (16 hpf), 88% (150 of 170) morphants presented with <12 somites compared with controls, and chevron/V-shaped somites normally observed beginning at this stage, a measure of developmental stage, were not observed in the morphants (Figure 3B). Therefore, 16-hpf morphants were developmentally equivalent to 14.5-hpf controls. By 24 hpf, morphants were noticeably smaller, and many structures exhibited a developmental delay compared with controls (91% [111 of 122] of 6-ng EGAP morphants presented with a similar phenotype; Figure 3C). Body length of morphants, a quantitative measure of developmental age after the 14-somite stage, was significantly decreased at 24 hpf compared with controls (Figure 3C). The majority of morphants exhibited pericardial and hindbrain edema by 48 hpf (Figure 3C), and by 4 to 5 dpf, severe pericardial edema was accompanied by apparent heart failure, likely causing lethality. To determine an effect on cell proliferation, controls and EGAP morphants were incubated with BrdUrd, fixed at 18 hpf, and BrdUrd incorporation determined by immunohistochemistry. Whereas controls showed extensive and ubiquitous cell proliferation at this stage, few BrdUrd-positive cells were present in morphants (Figure 3D). In contrast, significantly elevated numbers of apoptotic cells were detected in morphants (Figure 3E), indicating an essential role for EGAP in embryonic cell proliferation and survival. The EGAP MO-induced phenotype and lethality was rescued by overexpression of human cytomegalovirus immediate-early enhancer/promoter (CMV)-EGAP, verifying the observed phenotype was specific to EGAP depletion (Figure 3F). MO mediated depletion of zMak3 phenocopies the EGAP MO-induced phenotype (Figure 3B and 3C); combined with similar expression patterns and subunit assembly, these data are consistent with EGAP and Mak3 functioning together.
Because zEGAP and zMak3 mRNA levels were high in developing vascular structures (Figure 2), we used fli1-enhanced green fluoresent protein (EGFP) transgenic zebrafish to determine the role of the EGAP NAT in vessel development. Expression of fli1, an Ets domain family member, in the developing zebrafish begins early and is predominantly mesodermal but becomes restricted to differentiating angioblasts, endothelial cells, primitive blood cells, and pharyngeal arch mesenchyme, therefore serving as an early marker for endothelial cells.23–25 Control or EGAP-specific MO-injected Fli1-EGFP transgenic zebrafish25 were analyzed for EGFP expression at 15 and 26 hpf (Figure 4). EGFP fluorescence was detected throughout the anterior and posterior lateral mesoderm at 15 hpf in controls (Figure 4Ab). In contrast, morphants exhibited reduced EGFP fluorescence at 15 hpf (Figure 4Ac through 4Af), consistent with reduced cell proliferation at this time. Morphants exhibiting a phenotype (eg, reduced somite number; Figure 4BAc through 4Ae) expressed less EGFP compared with those that did not exhibit an obvious phenotype (Figure 4Af). At 26 hpf, EGFP expression in controls was similar to endogenous fli1 expression (Figure 4Ba through 4Bd).26 Patent dorsal aortae and posterior cardinal veins, with visible circulating blood cells, and well-formed segmental vessels were apparent in controls. In contrast, EGAP morphants exhibited less developed, disorganized vessels at 26 hpf (Figure 4Be through 4Bg). Major trunk vessels failed to form adequate lumens, and compared with controls, segmental vessels were truncated (Figure 4B, graph). Circulating blood cells present were often observed accumulated in proximal main trunk vessels, and little segmental vessel perfusion was observed even at later stages (data not shown). The morphant vessel phenotype was rescued by overexpression of CMV-EGAP (Figure 4C), implicating an essential role for the EGAP NAT in proper vessel development, likely through the regulation of vascular cell proliferation. Because EGAP is not restricted to the vasculature and cardiac failure was observed in morphants, we analyzed the developing heart to determine whether vessel defects are secondary to depressed cardiac development or function. We detected no obvious differences in heart rhythm at 24 and 48 hpf; both controls and morphants exhibited strong and coordinated contractility (data not shown) and little differences in heart rate (supplemental Figure 3A). ncx1h (sodium calcium exchanger) and cmcl2 (cardiac myosin light chain), two cardiac-specific differentiation markers,27,28 were expressed in normal temporal and spatial patterns in both controls and morphants (supplemental Figure 3B). These data suggest heart development and function are occurring normally, and heart failure occurs in response to primary defects in other tissues, including the vasculature.
Mechanism of Action of the EGAP NAT
A family of NATs catalyzes N-terminal acetylation of eukaryotic proteins, and each NAT recognizes distinct N-terminal sequences.12–15 In yeast, subclasses of proteins with Met-Ile, Met-Leu, Met-Trp, or Met-Phe N-termini are acetylated by yeast NatC/EGAP; NatC has the most limited number of identified protein substrates.14 To begin to define its mechanism of action, we focused on the mammalian TOR (mTOR) as a potential target of the EGAP NAT. TOR, a large serine/threonine protein kinase lying downstream of growth factor receptor signaling, is integral in coordinating cell growth and proliferation.29,30 Inactivation of Drosophila TOR results in reduced cell size and embryonic lethality.31 Similarly, mTOR null mouse embryos die shortly after implantation because of impaired cell proliferation, demonstrating mTOR is essential for embryonic development.32,33 mTOR activity is essential for vascular cell proliferation during vessel formation, and our previous data demonstrated rapid growth of embryonic SMCs is dependent on constitutive phosphatidylinositol 3-kinase-mTOR signaling events.34 Further, the TOR N termini is Met-Leu, providing the intriguing possibility that TOR is a direct EGAP NAT substrate. To determine whether the EGAP NAT regulates TOR function, control and EGAP morphants were analyzed at 24 hpf by whole-mount immunohistochemistry for total and phosphoS2448-zebrafish TOR (zTOR) levels. We found high and ubiquitous expression of total and phosphorylated zTOR in controls (Figure 5A). In contrast, both total and phosphoTOR were barely detectable in EGAP morphants (Figure 5A). Immunohistochemical data were verified by Western analysis of pooled controls or EGAP morphants (Figure 5B). In addition, phosphorylation of p70S6 kinase on T389, a site directly mediated by mTOR, was significantly reduced in EGAP morphants compared with controls (Figure 5B), strongly suggesting the proliferative defect in EGAP-deficient zebrafish embryos is, at least in part, mediated through decreased expression or signaling of zTOR.
To determine whether decreased TOR plays a role in mediating EGAP MO-induced growth defects, wild-type and fli-EGFP transgenic zebrafish embryos were incubated with the TOR inhibitor rapamycin. Compared with vehicle controls, rapamycin phenocopied EGAP morphant body length and vessel defects (Figure 6A). Low dose (10 nmol/L) resulted in similar defects as observed in EGAP morphants, whereas a higher dose (100 nmol/L) generated more severe defects, suggesting loss of TOR signaling is central to the observed EGAP-deficient phenotype. To confirm that loss of TOR signaling is involved in the EGAP MO-induced phenotype, we coinjected embryos with EGAP MO and constitutively active CMV-mTOR. Compared with EGAP MO injection alone, overexpression of mTOR in coinjected embryos rescued somite, body length, and vessel defects and lethality observed in EGAP morphants (Figure 6B). These data provide novel evidence that EGAP NAT activity is an essential regulator of TOR activity and, consequently, embryonic development.
Discussion
Rapid growth and proliferation of embryonic cells is essential for development of all organ systems and is responsible for postnatal/pathological injury repair processes. Regulation occurs through the coordinated expression and activity of a host of transcription factors, ligands and receptors, and intracellular signaling molecules. Therefore, it is necessary to understand both the mechanisms controlling gene expression and the post-translational modifications regulating the function of proteins involved in these processes. Through our studies to identify novel genes for which expression is high in the developing rat vasculature, absent in adult vessels, but increased in the setting of vascular injury, we cloned the vertebrate orthologues of the yeast NatC complex. Although many proteins are N-acetylated, there is only a small body of knowledge regarding the role of this protein modification in vertebrate systems, and virtually nothing is known of the functional role of the EGAP NAT during development. We show here that EGAP and Mak3 are expressed in overlapping temporal and spatial patterns during zebrafish development and that all three mammalian subunits specifically interact to form a stable, functional complex in vivo. To our knowledge, this is the first report showing the consequences of a lack of N-acetylation on vertebrate developmental processes. In particular, we are the first to show loss of EGAP/NatC-induced acetylation results in embryonic lethality marked, at least in part, by reduced cell proliferation and survival and attenuated vessel development, indicating EGAP NAT-induced acetylation is essential for embryonic development. In addition, we identified TOR as a downstream target of EGAP NAT activity. Reduced cell proliferation combined with increased cell death observed in EGAP morphants is consistent with cell growth and survival functions of the TOR signaling pathway, thus strongly implicating disruption of TOR signaling events as central to the proliferative defect in EGAP-deficient zebrafish embryos.
We first identified EGAP in rapidly proliferating vascular SMCs for which re-expression after vascular injury is associated with pathological neointima formation.11 In addition, another group showed EGAP expression correlates with cornea and skin maturation and re-epithelialization of wounded cornea, suggesting a role for EGAP NAT acetylation in epithelial development, differentiation, and wound healing.35 EGAP/Mak10p was first identified as a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae.36 Mak10p- and Mak3p-deficient yeast mutants fail to assemble the L-A dsRNA viral particle because of decreased acetylation of the killer viral major coat protein, Gag, and exhibit similar slow growth phenotypes on nonfermentable carbon sources.12,22 Only a few yeast NatC substrates have been identified, including the GTPase, Arl3p,37,38 and the mitochondrial proteins Kdg1p and Fum1p.22 Acetylation is necessary for recruitment to Golgi membranes for membrane trafficking (Arl3p) and for growth on nonfermentable carbon sources (Kdg1p and Fum1p). Our data expand on the role of the vertebrate complex, demonstrating it is essential for cell growth and survival and proper embryonic development at least in part through the regulation of TOR signaling events. Furthermore, functional subunit interactions and dynamic regulation of EGAP and Mak3 indicate control of this system occurs through the regulation of enzyme activity and EGAP NAT subunit expression. EGAP is expressed in several, but not all, tissues in the developing zebrafish, and EGAP NAT activity is likely essential for proliferative events associated with development of these tissues. Our data suggest a model whereby loss of EGAP NAT function causes primary defects in the developing vasculature and other affected tissues (eg, epidermis), resulting in a developmental delay, pericardial edema, and cardiac failure. We cannot definitively rule out the possibility that vascular abnormalities observed in the morphants are secondary events. However, because morphant hearts develop two contracting chambers, express differentiated markers in the correct pattern, and vessel defects are observed before heart abnormalities, it is likely that morphants exhibit primary vessel defects rather than secondary to cardiac dysfunction.
The EGAP/NatC complex and TOR proteins are evolutionarily conserved. Both are found in yeast, plants, worms, flies, and higher vertebrate species, and TOR is a principal regulator of embryonic development by controlling cell growth.39 Reduced TOR expression and signaling in EGAP-deficient zebrafish embryos combined with a similar phenotype after pharmacological inhibition of TOR and a rescued phenotype by TOR overexpression is compelling evidence that TOR is an important downstream target of EGAP NAT activity. Whether TOR is a direct EGAP NAT substrate has yet to be determined. The second amino acid residue of mTOR is leucine, an EGAP/NatC consensus sequence, suggesting this is a distinct possibility. Preliminary data in our laboratory show that EGAP NAT-rich systems express an acetylated form of TOR, whereas TOR acetylation is not observed in EGAP-deficient systems (data not shown). Alternatively, it is possible that TOR is not a direct EGAP NAT substrate but rather a downstream effector of an unidentified EGAP NAT substrate(s). Nevertheless, we were able to drive an almost complete rescue of the EGAP MO-induced phenotype with injection of constitutively active mTOR cDNA placing the TOR signaling pathway as a major target of this acetyltransferase. Our current studies are designed to determine whether TOR is a direct endogenous substrate and define the functional consequences of decreased TOR acetylation in EGAP NAT-deficient systems, which would add to the short list of proteins for which function is dependent on EGAP/NatC-induced acetylation and to define additional endogenous EGAP NAT substrates.
Recent findings indicate N-terminal acetylation is a highly conserved essential regulator of many biological processes, including embryonic growth control, vessel development, and SMC function. Distinct NAT mutant phenotypes, substrate specificity, and dynamically regulated subunit expression patterns indicate N-terminal acetylation of select protein substrates is a nonredundant, highly regulated controller of protein function. The available evidence suggests the EGAP NAT provides a novel regulatory role on a small subset of proteins, including TOR, essential for embryonic cell proliferation and growth. Identification of additional endogenous substrates and its role in pathophysiological processes will lend further insight into the biological significance of this acetyltransferase in higher vertebrate species and could serve as a fundamental platform for the design of a new class of pharmaceuticals useful in inhibiting a panel of proteins involved in pathological proliferative events.
Acknowledgments
This work was supported by a grant from the National Institutes of Health to M.C.M.W.-E. (R01HL063946). We thank Deborah Garrity for assistance with zebrafish image acquisition, expertise in zebrafish cardiac development and function, and cardiac-specific in situ hybridizations and Nicholas Barry for confocal image acquisition.
Footnotes
Original received September 30, 2005; revision received January 31, 2006; accepted February 8, 2006.
References
Schwartz SM. The intima: a new soil. Circ Res. 1999; 85: 877–879.
Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 84: 767–801.
Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Circ Res. 2005; 96: 280–291.
Cook CL, Weiser MC, Schwartz PE, Jones CL, Majack RA. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ Res. 1994; 74: 189–196.
Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev Biol. 1996; 178: 375–392. [Order article via Infotrieve]
Lee SH, Hungerford JE, Little CD, Iruela-Arispe ML. Proliferation and differentiation of smooth muscle cell precursors occurs simultaneously during the development of the vessel wall. Dev Dyn. 1997; 209: 342–352. [Order article via Infotrieve]
Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res. 1999; 36: 2–27. [Order article via Infotrieve]
Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003; 9: 685–693. [Order article via Infotrieve]
Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983; 49: 327–333. [Order article via Infotrieve]
Weiser-Evans MC, Quinn BE, Burkard MR, Stenmark KR. Transient reexpression of an embryonic autonomous growth phenotype by adult carotid artery smooth muscle cells after vascular injury. J Cell Physiol. 2000; 182: 12–23. [Order article via Infotrieve]
Weiser-Evans MC, Schwartz PE, Grieshaber NA, Belknap JK, Mourani PM, Majack RA, Stenmark KR. Novel embryonic genes are preferentially expressed by autonomously replicating rat embryonic and neointimal smooth muscle cells. Circ Res. 2000; 87: 608–615.
Polevoda B, Sherman F. Nalpha-terminal acetylation of eukaryotic proteins. J Biol Chem. 2000; 275: 36479–36482.
Polevoda B, Sherman F. Composition and function of the eukaryotic N-terminal acetyltransferase subunits. Biochem Biophys Res Commun. 2003; 308: 1–11. [Order article via Infotrieve]
Polevoda B, Sherman F. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. J Mol Biol. 2003; 325: 595–622. [Order article via Infotrieve]
Polevoda B, Norbeck J, Takakura H, Blomberg A, Sherman F. Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae. EMBO J. 1999; 18: 6155–6168. [Order article via Infotrieve]
Polevoda B, Cardillo TS, Doyle TC, Bedi GS, Sherman F. Nat3p and Mdm20p are required for function of yeast NatB Nalpha-terminal acetyltransferase and of actin and tropomyosin. J Biol Chem. 2003; 278: 30686–30697.
Singer JM, Shaw JM. Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin-actin interactions in budding yeast. Proc Natl Acad Sci U S A. 2003; 100: 7644–7649.
Gendron RL, Adams LC, Paradis H. Tubedown-1, a novel acetyltransferase associated with blood vessel development. Dev Dyn. 2000; 218: 300–315. [Order article via Infotrieve]
Choi SC, Chang JY, Han JK. A novel Xenopus acetyltransferase with a dynamic expression in early development. Biochem Biophys Res Commun. 2001; 285: 1338–1343. [Order article via Infotrieve]
Sugiura N, Adams SM, Corriveau RA. An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development. J Biol Chem. 2003; 278: 40113–40120.
Arnesen T, Anderson D, Baldersheim C, Lanotte M, Varhaug JE, Lillehaug JR. Identification and characterization of the human ARD1-NATH protein acetyltransferase complex. Biochem J. 2005; 386: 433–443. [Order article via Infotrieve]
Polevoda B, Sherman F. NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p. J Biol Chem. 2001; 276: 20154–20159.
Brown LA, Rodaway AR, Schilling TF, Jowett T, Ingham PW, Patient RK, Sharrocks AD. Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos. Mech Dev. 2000; 90: 237–252. [Order article via Infotrieve]
Sato Y. Role of ETS family transcription factors in vascular development and angiogenesis. Cell Struct Funct. 2001; 26: 19–24. [Order article via Infotrieve]
Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol. 2002; 248: 307–318. [Order article via Infotrieve]
Sprague J, Doerry E, Douglas S, Westerfield M. The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research. Nucleic Acids Res. 2001; 29: 87–90.
Ebert AM, Hume GL, Warren KS, Cook NP, Burns CG, Mohideen MA, Siegal G, Yelon D, Fishman MC, Garrity DM. Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts. Proc Natl Acad Sci U S A. 2005; 102: 17705–17710.
Yelon D, Ticho B, Halpern ME, Ruvinsky I, Ho RK, Silver LM, Stainier DY. The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development. 2000; 127: 2573–2582.
Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene. 2004; 23: 3151–3171. [Order article via Infotrieve]
Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004; 18: 1926–1945.
Zhang H, Stallock JP, Ng JC, Reinhard C, Neufeld TP. Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 2000; 14: 2712–2724.
Gangloff YG, Mueller M, Dann SG, Svoboda P, Sticker M, Spetz JF, Um SH, Brown EJ, Cereghini S, Thomas G, Kozma SC. Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development. Mol Cell Biol. 2004; 24: 9508–9516.
Murakami M, Ichisaka T, Maeda M, Oshiro N, Hara K, Edenhofer F, Kiyama H, Yonezawa K, Yamanaka S. mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol. 2004; 24: 6710–6718.
Mourani PM, Garl PJ, Wenzlau JM, Carpenter TC, Stenmark KR, Weiser-Evans MC. Unique, highly proliferative growth phenotype expressed by embryonic and neointimal smooth muscle cells is driven by constitutive Akt, mTOR, and p70S6K signaling and is actively repressed by PTEN. Circulation. 2004; 109: 1299–1306.
Yi XJ, Li XF, Yu FS. A novel epithelial wound-related gene is abundantly expressed in developing rat cornea and skin. Curr Eye Res. 2000; 20: 430–440. [Order article via Infotrieve]
Lee YJ, Wickner RB. MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs. Genetics. 1992; 132: 87–96.
Behnia R, Panic B, Whyte JR, Munro S. Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p. Nat Cell Biol. 2004; 6: 405–413. [Order article via Infotrieve]
Setty SR, Strochlic TI, Tong AH, Boone C, Burd CG. Golgi targeting of ARF-like GTPase Arl3p requires its Nalpha-acetylation and the integral membrane protein Sys1p. Nat Cell Biol. 2004; 6: 414–419. [Order article via Infotrieve]
Jacinto E, Hall MN. Tor signaling in bugs, brain and brawn. Nat Rev Mol Cell Biol. 2003; 4: 117–126. [Order article via Infotrieve]
the Departments of Renal Medicine (R.A.N., M.C.M.W.-E.), Cell and Developmental Biology (K.B.A., M.C.M.W.-E.), Craniofacial Biology (P.S., K.B.A.), Pediatrics (J.M.W., P.J.G., K.R.S., M.C.M.W.-E.)
Cardiovascular Pulmonary Research (J.W.), University of Colorado Health Sciences Center, Denver.
Abstract
N-terminal protein acetylation, catalyzed by N-terminal acetyltransferases (NATs) recognizing distinct N-terminal sequences, is gaining recognition as an essential regulator of normal cell function, but little is known of its role in vertebrate development. We previously cloned a novel gene, embryonic growth-associated protein (EGAP), the expression of which is associated with rapid vascular smooth muscle cell proliferation during development. We show herein EGAP is the mammalian/zebrafish homologue of yeast Mak10p, one subunit of the yeast NatC complex, and describe the cloning of its binding partners Mak3 and Mak31. The EGAP NAT forms a functional complex in mammalian cells, is evolutionarily conserved, and developmentally regulated. It is widely but not ubiquitously expressed during early zebrafish development but undetectable in later developmental stages. We demonstrate EGAP- and Mak3-deficient zebrafish fail to develop because of, in part, decreased cell proliferation, increased apoptosis, and poor blood vessel formation contributing to embryonic lethality. We examined the role of target of rapamycin (TOR), a highly conserved protein kinase controlling cell growth, as a physiological target of EGAP NAT acetylation. Compared with controls, TOR expression and signaling is significantly reduced in EGAP morphants. Pharmacological inhibition of TOR with rapamycin phenocopied the EGAP morpholino oligonucleotide-induced growth and vessel defects. Overexpression of constitutively active TOR rescued EGAP morphants, suggesting TOR is a direct or indirect endogenous substrate of the EGAP NAT complex. These data suggest the EGAP NAT complex is an essential regulatory enzyme controlling the function of a subset of proteins required for embryonic growth control and vessel development.
Key Words: EGAP TOR N-terminal acetyltransferase vascular development vascular cell proliferation
Introduction
Smooth muscle cell (SMC) proliferation plays a prominent role in normal vessel development and in vascular pathologies, including atherosclerosis and restenosis after angioplasty and stent placement.1–3 During vessel development, SMCs undergo a transient phase of rapid proliferation during which the vessel wall acquires its complement of SMCs.4–8 Replication decreases with vessel maturation as SMCs reach a highly quiescent, differentiated state in adult vessels. However, a hallmark of vascular occlusive disease and interventions in response to disease is a transient increase in SMC replication to levels similar to those exhibited during embryonic life.9,10 SMCs cultured from embryonic vessels or from the neointima of injured vessels during periods of rapid in vivo growth exhibit a unique growth phenotype characterized by rapid serum-stimulated growth and the ability to replicate in a mitogen-independent manner.4,10 This is distinct from SMCs derived from adult uninjured vessels, which exhibit slower serum-stimulated growth and become quiescent under mitogen-deprived conditions. However, the mechanisms regulating this unique SMC growth phenotype at these times have yet to be fully elucidated.
To facilitate our characterization of this rapid proliferative phenotype, we previously cloned several novel genes selectively expressed by rapidly proliferating cells during early vascular development and only re-expressed in the adult vasculature by cells responding to pathological injury.11 One of these genes, which we named embryonic growth-associated protein (EGAP), is expressed in a variety of tissues in the developing rat embryo but restricted in expression in the adult, remaining detectable only in tissues undergoing continual cell renewal or in cells responding to pathological injury. We report EGAP, the vertebrate orthologue of the yeast Mak10p protein, is one subunit of a novel N-terminal acetyltransferase (NAT) that is highly conserved among vertebrate species.
N-terminal acetylation is a post-translational modification of proteins in which the acetyl group of AcetylCoA is transferred to the initiator methionine or most N-terminal amino acid.12–14 This irreversible protein modification is catalyzed by a family of NATs, each recognizing distinct N-termini sequences of specific protein substrates. Most information regarding N-acetylation has been obtained from studies in yeast, which express at least three NATs: NatA, NatB, and NatC.12,14,15 Each complex consists of independently transcribed subunits; loss or mutation of any one subunit results in loss of NAT activity. N-acetylation may alter protein functions such as protein stability, protein-protein interactions, further modifications such as phosphorylation, and translocation to specific cellular compartments.12 For instance, yeast NatB acetylation of actin and tropomyosin is essential for interactions of these proteins and actin filament stabilization.16,17 Although many proteins are N-terminal acetylated, there is only a small body of knowledge regarding the role of this protein modification in vertebrate systems. Existing information focuses on the role of the vertebrate Ard1–NatH complex, the yeast NatA-related complex that has the largest number of endogenous substrates identified to date.18–21 In contrast, much less is known of the NatC complex, consisting of Mak3p, Mak10p, and Mak31p, or its endogenous substrates.22 Subunit copurification and similar slow growth phenotypes of all three yeast deletion strains (mak3p-, mak10p-, and mak31p-) highly suggest that interaction of subunits is required for NatC enzyme activity. However, virtually nothing is known of the biological significance of the EGAP/NatC complex in vertebrate species. Our data establish the EGAP NAT complex functionally coassembles in mammalian cells to regulate cell proliferation and is essential for embryonic development, at least in part through the regulation of target of rapamycin (TOR) signaling events.
Materials and Methods
An expanded Materials and Methods section can be found in the online supplement, available at http://circres.ahajournals.org.
Genomic Sequence Analysis and Morpholino Design
A positive mouse bacterial artificial chromosome (BAC) clone corresponding to rat EGAP cDNA sequence was obtained from Research Genetics (mouse RPCI BAC 465-E7) and mapped to deduce the genomic structure and complete cDNA sequence. Full-length Flag-, Myc-, and influenza hemagglutinin (HA)-tagged expression constructs were generated for mouse Mak31, EGAP, and Mak3, respectively, by RT-PCR using embryonic day 14 mouse poly(A+) RNA. The genomic organization of the zebrafish EGAP and Mak3 loci were deduced from comparisons of their cDNA sequences with genomic trace sequences using the BLAST program on the NCBI website. Antisense morpholino oligonucleotides (MOs) were designed against the splice donor at the end of the first coding exons.
In Situ Hybridization, Immunohistochemistry, and TUNEL Staining
Whole-mount in situ hybridization was performed as described previously. Whole-mount immunohistochemistry was performed using the Vectastain Avidin/Biotin/Horseradish peroxidase ABC Elite System as described previously. TUNEL was used to assess apoptosis in control and EGAP morphants. Briefly, embryos were permeabilized, washed, incubated in terminal transferase (Roche), and biotin-labeled ddUTP (Roche) and biotin incorporation detected with the peroxidase ABC kit (Vector) using diaminobenzidine as chromogen. For 5-bromodeoxyuridine (BrdUrd) immunohistochemistry, embryos were incubated with BrdUrd, and 5-μm cryosections were immunohistochemically stained for BrdUrd as described previously.4
Results
Identification and Functional Role of the EGAP NAT Complex
Verification that EGAP mRNA is developmentally regulated in rat aortic SMCs and sequence analysis showing the EGAP NAT complex is highly conserved can be found in supplemental Figures 1 and 2, available online at http://circres.ahajournals.org.
Studies from yeast proposed NatC subunit expression and assembly are required for its acetyltransferase activity.22 Thus, we used coexpression and coimmunoprecipitation analyses to determine whether the mammalian complex similarly colocalizes and functionally assembles in vivo. Human embryonic kidney 293 (HEK 293) cells were transiently transfected with Myc-tagged mouse EGAP (mEGAP), HA-tagged mMak3, and Flag-tagged mMak31 expression constructs to determine cellular localization and to determine overlap in cellular distribution (Figure 1A). All proteins appear in the cytoplasm, are excluded from the nucleus, and show a high degree of overlap in cellular distribution (Figure 1A, merge). To determine whether the mammalian proteins coassemble in living cells, whole cell lysates from transfected HEK 293 cells were immunoprecipitated using anti-Myc or anti-Flag antibodies, and Western blotting was performed using anti-Myc, anti-Flag, or anti-HA antibodies (Figure 1B). Myc-mEGAP, HA-mMak3, and Flag-mMak31 were detected in cells overexpressing all three subunits using either anti-Myc or anti-Flag for immunoprecipitation (Figure 1B, lanes 1 and 8), demonstrating mEGAP, mMak3, and mMak31 coassemble in mammalian cells. The results from these reciprocal immunoprecipitation experiments strongly suggest formation of a stable in vivo complex in mammalian cells. Omission of mMak31 (Figure 1B, lanes 3 and 9), mMak3 (Figure 1B, lanes 4 and 10), or mEGAP (Figure 1B, lanes 5 and 11) from transfections resulted in loss of subunit assembly, especially recruitment and association of mMak3, the catalytic subunit, with mEGAP and mMak31 (mEGAP and mMak31 continued to associate in the absence of mMak3; Figure 1B, lanes 4 and 10).
Because the mammalian EGAP NAT proteins form a stable in vivo complex and EGAP is associated with rapid cell proliferation,11 we determined the functional role of subunit assembly on cell growth. Transfected HEK 293 cells were incubated in low serum media in the presence of BrdUrd for 24 hours, and the degree of BrdUrd incorporation, and therefore DNA synthesis, was determined. Compared with nontransfected cells that begin to exhibit a decline in replication after 24 hours in low serum, cells overexpressing all three subunits maintained high rates of BrdUrd incorporation (Figure 1C). Omission of mEGAP, mMak3, or mMak31 from transfections resulted in reduced levels of BrdUrd incorporation (similar to nontransfected cells), highly suggestive of a functional role for the EGAP NAT complex in regulating cell proliferation.
The EGAP NAT Complex Is Essential for Zebrafish Development
Our previous data11 suggested expression and subsequent downregulation of EGAP plays an important role in development and differentiation, including vascular cell growth and differentiation. We next used the zebrafish model to determine the biological significance of the EGAP NAT during development. Using whole-mount in situ hybridization, we found zEGAP mRNA is expressed at high levels in the developing zebrafish embryo as early as 6 hours post fertilization (hpf) and remains high in many developing tissues through 18 hpf and 28 hpf (Figure 2Aa through 2Ac). Consistent with our previous studies in the rat, zEGAP expression was high in developing vascular structures, implicating an important role in blood vessel development (Figure 2Ad through 2Ag). Staining was also strong in the epidermis but not the neural tube or notochord (Figure 2Ag). Staining becomes restricted to the developing head by 48 hpf (Figure 2Ah) and by 5 days post fertilization (dpf), is observed only in the epidermis (Figure 2Ai), where actively replicating cells were detected (Figure 2Aj). The temporal and spatial pattern of zMak3 expression is very similar to that of zEGAP (Figure 2B), and both are similar to those we reported previously for EGAP in the developing rat; thus, EGAP and Mak3 are evolutionarily conserved, developmentally regulated genes.
To establish the EGAP NAT is essential for zebrafish development, we diminished EGAP expression using MOs targeted to the splice donor site at the end of EGAP exon 2 (containing the ATG start codon). Microinjection of 6 ng MO into zebrafish embryos produced a truncated form of EGAP through insertion of a partial intron sequence (Figure 3A). Compared with control MO-injected embryos, EGAP morphants exhibited significant delays in growth (Figure 3B and 3C), and very few live EGAP morphants were observed after 4 to 5 dpf. At the 14-somite stage (16 hpf), 88% (150 of 170) morphants presented with <12 somites compared with controls, and chevron/V-shaped somites normally observed beginning at this stage, a measure of developmental stage, were not observed in the morphants (Figure 3B). Therefore, 16-hpf morphants were developmentally equivalent to 14.5-hpf controls. By 24 hpf, morphants were noticeably smaller, and many structures exhibited a developmental delay compared with controls (91% [111 of 122] of 6-ng EGAP morphants presented with a similar phenotype; Figure 3C). Body length of morphants, a quantitative measure of developmental age after the 14-somite stage, was significantly decreased at 24 hpf compared with controls (Figure 3C). The majority of morphants exhibited pericardial and hindbrain edema by 48 hpf (Figure 3C), and by 4 to 5 dpf, severe pericardial edema was accompanied by apparent heart failure, likely causing lethality. To determine an effect on cell proliferation, controls and EGAP morphants were incubated with BrdUrd, fixed at 18 hpf, and BrdUrd incorporation determined by immunohistochemistry. Whereas controls showed extensive and ubiquitous cell proliferation at this stage, few BrdUrd-positive cells were present in morphants (Figure 3D). In contrast, significantly elevated numbers of apoptotic cells were detected in morphants (Figure 3E), indicating an essential role for EGAP in embryonic cell proliferation and survival. The EGAP MO-induced phenotype and lethality was rescued by overexpression of human cytomegalovirus immediate-early enhancer/promoter (CMV)-EGAP, verifying the observed phenotype was specific to EGAP depletion (Figure 3F). MO mediated depletion of zMak3 phenocopies the EGAP MO-induced phenotype (Figure 3B and 3C); combined with similar expression patterns and subunit assembly, these data are consistent with EGAP and Mak3 functioning together.
Because zEGAP and zMak3 mRNA levels were high in developing vascular structures (Figure 2), we used fli1-enhanced green fluoresent protein (EGFP) transgenic zebrafish to determine the role of the EGAP NAT in vessel development. Expression of fli1, an Ets domain family member, in the developing zebrafish begins early and is predominantly mesodermal but becomes restricted to differentiating angioblasts, endothelial cells, primitive blood cells, and pharyngeal arch mesenchyme, therefore serving as an early marker for endothelial cells.23–25 Control or EGAP-specific MO-injected Fli1-EGFP transgenic zebrafish25 were analyzed for EGFP expression at 15 and 26 hpf (Figure 4). EGFP fluorescence was detected throughout the anterior and posterior lateral mesoderm at 15 hpf in controls (Figure 4Ab). In contrast, morphants exhibited reduced EGFP fluorescence at 15 hpf (Figure 4Ac through 4Af), consistent with reduced cell proliferation at this time. Morphants exhibiting a phenotype (eg, reduced somite number; Figure 4BAc through 4Ae) expressed less EGFP compared with those that did not exhibit an obvious phenotype (Figure 4Af). At 26 hpf, EGFP expression in controls was similar to endogenous fli1 expression (Figure 4Ba through 4Bd).26 Patent dorsal aortae and posterior cardinal veins, with visible circulating blood cells, and well-formed segmental vessels were apparent in controls. In contrast, EGAP morphants exhibited less developed, disorganized vessels at 26 hpf (Figure 4Be through 4Bg). Major trunk vessels failed to form adequate lumens, and compared with controls, segmental vessels were truncated (Figure 4B, graph). Circulating blood cells present were often observed accumulated in proximal main trunk vessels, and little segmental vessel perfusion was observed even at later stages (data not shown). The morphant vessel phenotype was rescued by overexpression of CMV-EGAP (Figure 4C), implicating an essential role for the EGAP NAT in proper vessel development, likely through the regulation of vascular cell proliferation. Because EGAP is not restricted to the vasculature and cardiac failure was observed in morphants, we analyzed the developing heart to determine whether vessel defects are secondary to depressed cardiac development or function. We detected no obvious differences in heart rhythm at 24 and 48 hpf; both controls and morphants exhibited strong and coordinated contractility (data not shown) and little differences in heart rate (supplemental Figure 3A). ncx1h (sodium calcium exchanger) and cmcl2 (cardiac myosin light chain), two cardiac-specific differentiation markers,27,28 were expressed in normal temporal and spatial patterns in both controls and morphants (supplemental Figure 3B). These data suggest heart development and function are occurring normally, and heart failure occurs in response to primary defects in other tissues, including the vasculature.
Mechanism of Action of the EGAP NAT
A family of NATs catalyzes N-terminal acetylation of eukaryotic proteins, and each NAT recognizes distinct N-terminal sequences.12–15 In yeast, subclasses of proteins with Met-Ile, Met-Leu, Met-Trp, or Met-Phe N-termini are acetylated by yeast NatC/EGAP; NatC has the most limited number of identified protein substrates.14 To begin to define its mechanism of action, we focused on the mammalian TOR (mTOR) as a potential target of the EGAP NAT. TOR, a large serine/threonine protein kinase lying downstream of growth factor receptor signaling, is integral in coordinating cell growth and proliferation.29,30 Inactivation of Drosophila TOR results in reduced cell size and embryonic lethality.31 Similarly, mTOR null mouse embryos die shortly after implantation because of impaired cell proliferation, demonstrating mTOR is essential for embryonic development.32,33 mTOR activity is essential for vascular cell proliferation during vessel formation, and our previous data demonstrated rapid growth of embryonic SMCs is dependent on constitutive phosphatidylinositol 3-kinase-mTOR signaling events.34 Further, the TOR N termini is Met-Leu, providing the intriguing possibility that TOR is a direct EGAP NAT substrate. To determine whether the EGAP NAT regulates TOR function, control and EGAP morphants were analyzed at 24 hpf by whole-mount immunohistochemistry for total and phosphoS2448-zebrafish TOR (zTOR) levels. We found high and ubiquitous expression of total and phosphorylated zTOR in controls (Figure 5A). In contrast, both total and phosphoTOR were barely detectable in EGAP morphants (Figure 5A). Immunohistochemical data were verified by Western analysis of pooled controls or EGAP morphants (Figure 5B). In addition, phosphorylation of p70S6 kinase on T389, a site directly mediated by mTOR, was significantly reduced in EGAP morphants compared with controls (Figure 5B), strongly suggesting the proliferative defect in EGAP-deficient zebrafish embryos is, at least in part, mediated through decreased expression or signaling of zTOR.
To determine whether decreased TOR plays a role in mediating EGAP MO-induced growth defects, wild-type and fli-EGFP transgenic zebrafish embryos were incubated with the TOR inhibitor rapamycin. Compared with vehicle controls, rapamycin phenocopied EGAP morphant body length and vessel defects (Figure 6A). Low dose (10 nmol/L) resulted in similar defects as observed in EGAP morphants, whereas a higher dose (100 nmol/L) generated more severe defects, suggesting loss of TOR signaling is central to the observed EGAP-deficient phenotype. To confirm that loss of TOR signaling is involved in the EGAP MO-induced phenotype, we coinjected embryos with EGAP MO and constitutively active CMV-mTOR. Compared with EGAP MO injection alone, overexpression of mTOR in coinjected embryos rescued somite, body length, and vessel defects and lethality observed in EGAP morphants (Figure 6B). These data provide novel evidence that EGAP NAT activity is an essential regulator of TOR activity and, consequently, embryonic development.
Discussion
Rapid growth and proliferation of embryonic cells is essential for development of all organ systems and is responsible for postnatal/pathological injury repair processes. Regulation occurs through the coordinated expression and activity of a host of transcription factors, ligands and receptors, and intracellular signaling molecules. Therefore, it is necessary to understand both the mechanisms controlling gene expression and the post-translational modifications regulating the function of proteins involved in these processes. Through our studies to identify novel genes for which expression is high in the developing rat vasculature, absent in adult vessels, but increased in the setting of vascular injury, we cloned the vertebrate orthologues of the yeast NatC complex. Although many proteins are N-acetylated, there is only a small body of knowledge regarding the role of this protein modification in vertebrate systems, and virtually nothing is known of the functional role of the EGAP NAT during development. We show here that EGAP and Mak3 are expressed in overlapping temporal and spatial patterns during zebrafish development and that all three mammalian subunits specifically interact to form a stable, functional complex in vivo. To our knowledge, this is the first report showing the consequences of a lack of N-acetylation on vertebrate developmental processes. In particular, we are the first to show loss of EGAP/NatC-induced acetylation results in embryonic lethality marked, at least in part, by reduced cell proliferation and survival and attenuated vessel development, indicating EGAP NAT-induced acetylation is essential for embryonic development. In addition, we identified TOR as a downstream target of EGAP NAT activity. Reduced cell proliferation combined with increased cell death observed in EGAP morphants is consistent with cell growth and survival functions of the TOR signaling pathway, thus strongly implicating disruption of TOR signaling events as central to the proliferative defect in EGAP-deficient zebrafish embryos.
We first identified EGAP in rapidly proliferating vascular SMCs for which re-expression after vascular injury is associated with pathological neointima formation.11 In addition, another group showed EGAP expression correlates with cornea and skin maturation and re-epithelialization of wounded cornea, suggesting a role for EGAP NAT acetylation in epithelial development, differentiation, and wound healing.35 EGAP/Mak10p was first identified as a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae.36 Mak10p- and Mak3p-deficient yeast mutants fail to assemble the L-A dsRNA viral particle because of decreased acetylation of the killer viral major coat protein, Gag, and exhibit similar slow growth phenotypes on nonfermentable carbon sources.12,22 Only a few yeast NatC substrates have been identified, including the GTPase, Arl3p,37,38 and the mitochondrial proteins Kdg1p and Fum1p.22 Acetylation is necessary for recruitment to Golgi membranes for membrane trafficking (Arl3p) and for growth on nonfermentable carbon sources (Kdg1p and Fum1p). Our data expand on the role of the vertebrate complex, demonstrating it is essential for cell growth and survival and proper embryonic development at least in part through the regulation of TOR signaling events. Furthermore, functional subunit interactions and dynamic regulation of EGAP and Mak3 indicate control of this system occurs through the regulation of enzyme activity and EGAP NAT subunit expression. EGAP is expressed in several, but not all, tissues in the developing zebrafish, and EGAP NAT activity is likely essential for proliferative events associated with development of these tissues. Our data suggest a model whereby loss of EGAP NAT function causes primary defects in the developing vasculature and other affected tissues (eg, epidermis), resulting in a developmental delay, pericardial edema, and cardiac failure. We cannot definitively rule out the possibility that vascular abnormalities observed in the morphants are secondary events. However, because morphant hearts develop two contracting chambers, express differentiated markers in the correct pattern, and vessel defects are observed before heart abnormalities, it is likely that morphants exhibit primary vessel defects rather than secondary to cardiac dysfunction.
The EGAP/NatC complex and TOR proteins are evolutionarily conserved. Both are found in yeast, plants, worms, flies, and higher vertebrate species, and TOR is a principal regulator of embryonic development by controlling cell growth.39 Reduced TOR expression and signaling in EGAP-deficient zebrafish embryos combined with a similar phenotype after pharmacological inhibition of TOR and a rescued phenotype by TOR overexpression is compelling evidence that TOR is an important downstream target of EGAP NAT activity. Whether TOR is a direct EGAP NAT substrate has yet to be determined. The second amino acid residue of mTOR is leucine, an EGAP/NatC consensus sequence, suggesting this is a distinct possibility. Preliminary data in our laboratory show that EGAP NAT-rich systems express an acetylated form of TOR, whereas TOR acetylation is not observed in EGAP-deficient systems (data not shown). Alternatively, it is possible that TOR is not a direct EGAP NAT substrate but rather a downstream effector of an unidentified EGAP NAT substrate(s). Nevertheless, we were able to drive an almost complete rescue of the EGAP MO-induced phenotype with injection of constitutively active mTOR cDNA placing the TOR signaling pathway as a major target of this acetyltransferase. Our current studies are designed to determine whether TOR is a direct endogenous substrate and define the functional consequences of decreased TOR acetylation in EGAP NAT-deficient systems, which would add to the short list of proteins for which function is dependent on EGAP/NatC-induced acetylation and to define additional endogenous EGAP NAT substrates.
Recent findings indicate N-terminal acetylation is a highly conserved essential regulator of many biological processes, including embryonic growth control, vessel development, and SMC function. Distinct NAT mutant phenotypes, substrate specificity, and dynamically regulated subunit expression patterns indicate N-terminal acetylation of select protein substrates is a nonredundant, highly regulated controller of protein function. The available evidence suggests the EGAP NAT provides a novel regulatory role on a small subset of proteins, including TOR, essential for embryonic cell proliferation and growth. Identification of additional endogenous substrates and its role in pathophysiological processes will lend further insight into the biological significance of this acetyltransferase in higher vertebrate species and could serve as a fundamental platform for the design of a new class of pharmaceuticals useful in inhibiting a panel of proteins involved in pathological proliferative events.
Acknowledgments
This work was supported by a grant from the National Institutes of Health to M.C.M.W.-E. (R01HL063946). We thank Deborah Garrity for assistance with zebrafish image acquisition, expertise in zebrafish cardiac development and function, and cardiac-specific in situ hybridizations and Nicholas Barry for confocal image acquisition.
Footnotes
Original received September 30, 2005; revision received January 31, 2006; accepted February 8, 2006.
References
Schwartz SM. The intima: a new soil. Circ Res. 1999; 85: 877–879.
Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 84: 767–801.
Yoshida T, Owens GK. Molecular determinants of vascular smooth muscle cell diversity. Circ Res. 2005; 96: 280–291.
Cook CL, Weiser MC, Schwartz PE, Jones CL, Majack RA. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ Res. 1994; 74: 189–196.
Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the aortic vessel wall as defined by vascular smooth muscle and extracellular matrix markers. Dev Biol. 1996; 178: 375–392. [Order article via Infotrieve]
Lee SH, Hungerford JE, Little CD, Iruela-Arispe ML. Proliferation and differentiation of smooth muscle cell precursors occurs simultaneously during the development of the vessel wall. Dev Dyn. 1997; 209: 342–352. [Order article via Infotrieve]
Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res. 1999; 36: 2–27. [Order article via Infotrieve]
Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003; 9: 685–693. [Order article via Infotrieve]
Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983; 49: 327–333. [Order article via Infotrieve]
Weiser-Evans MC, Quinn BE, Burkard MR, Stenmark KR. Transient reexpression of an embryonic autonomous growth phenotype by adult carotid artery smooth muscle cells after vascular injury. J Cell Physiol. 2000; 182: 12–23. [Order article via Infotrieve]
Weiser-Evans MC, Schwartz PE, Grieshaber NA, Belknap JK, Mourani PM, Majack RA, Stenmark KR. Novel embryonic genes are preferentially expressed by autonomously replicating rat embryonic and neointimal smooth muscle cells. Circ Res. 2000; 87: 608–615.
Polevoda B, Sherman F. Nalpha-terminal acetylation of eukaryotic proteins. J Biol Chem. 2000; 275: 36479–36482.
Polevoda B, Sherman F. Composition and function of the eukaryotic N-terminal acetyltransferase subunits. Biochem Biophys Res Commun. 2003; 308: 1–11. [Order article via Infotrieve]
Polevoda B, Sherman F. N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins. J Mol Biol. 2003; 325: 595–622. [Order article via Infotrieve]
Polevoda B, Norbeck J, Takakura H, Blomberg A, Sherman F. Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisiae. EMBO J. 1999; 18: 6155–6168. [Order article via Infotrieve]
Polevoda B, Cardillo TS, Doyle TC, Bedi GS, Sherman F. Nat3p and Mdm20p are required for function of yeast NatB Nalpha-terminal acetyltransferase and of actin and tropomyosin. J Biol Chem. 2003; 278: 30686–30697.
Singer JM, Shaw JM. Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin-actin interactions in budding yeast. Proc Natl Acad Sci U S A. 2003; 100: 7644–7649.
Gendron RL, Adams LC, Paradis H. Tubedown-1, a novel acetyltransferase associated with blood vessel development. Dev Dyn. 2000; 218: 300–315. [Order article via Infotrieve]
Choi SC, Chang JY, Han JK. A novel Xenopus acetyltransferase with a dynamic expression in early development. Biochem Biophys Res Commun. 2001; 285: 1338–1343. [Order article via Infotrieve]
Sugiura N, Adams SM, Corriveau RA. An evolutionarily conserved N-terminal acetyltransferase complex associated with neuronal development. J Biol Chem. 2003; 278: 40113–40120.
Arnesen T, Anderson D, Baldersheim C, Lanotte M, Varhaug JE, Lillehaug JR. Identification and characterization of the human ARD1-NATH protein acetyltransferase complex. Biochem J. 2005; 386: 433–443. [Order article via Infotrieve]
Polevoda B, Sherman F. NatC Nalpha-terminal acetyltransferase of yeast contains three subunits, Mak3p, Mak10p, and Mak31p. J Biol Chem. 2001; 276: 20154–20159.
Brown LA, Rodaway AR, Schilling TF, Jowett T, Ingham PW, Patient RK, Sharrocks AD. Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos. Mech Dev. 2000; 90: 237–252. [Order article via Infotrieve]
Sato Y. Role of ETS family transcription factors in vascular development and angiogenesis. Cell Struct Funct. 2001; 26: 19–24. [Order article via Infotrieve]
Lawson ND, Weinstein BM. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol. 2002; 248: 307–318. [Order article via Infotrieve]
Sprague J, Doerry E, Douglas S, Westerfield M. The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research. Nucleic Acids Res. 2001; 29: 87–90.
Ebert AM, Hume GL, Warren KS, Cook NP, Burns CG, Mohideen MA, Siegal G, Yelon D, Fishman MC, Garrity DM. Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts. Proc Natl Acad Sci U S A. 2005; 102: 17705–17710.
Yelon D, Ticho B, Halpern ME, Ruvinsky I, Ho RK, Silver LM, Stainier DY. The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development. Development. 2000; 127: 2573–2582.
Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene. 2004; 23: 3151–3171. [Order article via Infotrieve]
Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004; 18: 1926–1945.
Zhang H, Stallock JP, Ng JC, Reinhard C, Neufeld TP. Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 2000; 14: 2712–2724.
Gangloff YG, Mueller M, Dann SG, Svoboda P, Sticker M, Spetz JF, Um SH, Brown EJ, Cereghini S, Thomas G, Kozma SC. Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development. Mol Cell Biol. 2004; 24: 9508–9516.
Murakami M, Ichisaka T, Maeda M, Oshiro N, Hara K, Edenhofer F, Kiyama H, Yonezawa K, Yamanaka S. mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol. 2004; 24: 6710–6718.
Mourani PM, Garl PJ, Wenzlau JM, Carpenter TC, Stenmark KR, Weiser-Evans MC. Unique, highly proliferative growth phenotype expressed by embryonic and neointimal smooth muscle cells is driven by constitutive Akt, mTOR, and p70S6K signaling and is actively repressed by PTEN. Circulation. 2004; 109: 1299–1306.
Yi XJ, Li XF, Yu FS. A novel epithelial wound-related gene is abundantly expressed in developing rat cornea and skin. Curr Eye Res. 2000; 20: 430–440. [Order article via Infotrieve]
Lee YJ, Wickner RB. MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs. Genetics. 1992; 132: 87–96.
Behnia R, Panic B, Whyte JR, Munro S. Targeting of the Arf-like GTPase Arl3p to the Golgi requires N-terminal acetylation and the membrane protein Sys1p. Nat Cell Biol. 2004; 6: 405–413. [Order article via Infotrieve]
Setty SR, Strochlic TI, Tong AH, Boone C, Burd CG. Golgi targeting of ARF-like GTPase Arl3p requires its Nalpha-acetylation and the integral membrane protein Sys1p. Nat Cell Biol. 2004; 6: 414–419. [Order article via Infotrieve]
Jacinto E, Hall MN. Tor signaling in bugs, brain and brawn. Nat Rev Mol Cell Biol. 2003; 4: 117–126. [Order article via Infotrieve]
您现在查看是摘要介绍页，详见ORG附件。