From the Department of Microbiology and
Department of Molecular and Cellular Physiology, Health Sciences
Center, University of Virginia, Charlottesville, Virginia 22908
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rho-related GTP-binding proteins constitute a functionally distinct group within the small molecular weight (smw)1 G protein family, which includes Rho A, B, C, and G, Rac1 and Rac2, Cdc42, and TC10 (1). These proteins are 30% identical to Ras and 55% identical to each other. The Rho family members have been linked to a variety of cellular effects, including changes in cytoskeletal dynamics (actin polymerization and reorganization), gene expression (p38/Jun NH2-terminal kinase and serum response factor activity), cleavage furrow formation, G1 cell cycle progression, endocytosis, exocytosis, and superoxide production (2).
Regulation of the cytoskeleton by Rho family members seems to be
dependent on a cascade of events within the actinomyosin system (2). In
response to a variety of stimuli (including bradykinin, tumor necrosis
factor-, and interleukin-1) cells send out exploratory filopodia, a
process regulated by Cdc42. Activation of Rac (by Cdc42-mediated
signaling or in response to stimulation with platelet-derived growth
factor, EGF, bombeson, or insulin) results in extensions of broad
sheet-like lamellipodia. Finally, activation of Rho (by Rac-mediated
signaling or by stimulation with bombesin or lysophosphatidic acid)
results in the formation of actin stress fibers and focal adhesions
(3-8). Each of these structures is formed by the ordered
polymerization of F-actin and presumably serve to mediate specialized
cellular functions, such as endocytosis, cell migration, and cell
division (9).
Like all smw G proteins, Rho family members exist in an inactive GDP-bound form and an active GTP-bound form. The rate of conversion between the GDP-bound form and GTP bound form is modulated by guanine nucleotide dissociation inhibitors, which inhibit GDP dissociation, guanine nucleotide exchange factors (GEFs), which stimulate the replacement of GDP by GTP, and by GTPase-activating proteins (GAPs), which stimulate the rate of intrinsic GTP hydrolysis by the GTPase (10). To date, at least seven mammalian GEFs and 10 mammalian GAPs have been identified for the Rho GTPase family (11-14) The existence of multiple GAP proteins suggests that these proteins may act on different subcellular pools of Rho proteins or may have different effector-functions upon association with Rho proteins. Although GAP proteins down-regulate the signal input by rapidly converting the G protein to its inactive GDP bound state, certain GAP proteins, including p120Ras GAP, n-chimaerin, and phospholipase C (a GAP for heterotrimeric G proteins) simultaneously send a signal that is required for downstream signaling from the G protein (15-17). For example, microinjection of the Rac GAP, n-chimaerin, (like activated Rac) induces the formation of lamellipodia, an effect that is dependent on Rac, but not dependent on the GAP activity of n-chimaerin (16). Apparently, the binding of n-chimaerin to activated Rac enhances a functional interaction between n-chimaerin and other unknown protein(s) to regulate cellular morphology.
It is currently unclear how the Rho cascade is activated and how the signal progresses from one smw G protein to another. It is possible that one smw G protein may alter the GEF or GAP activity toward another smw G protein to modify signal transduction. Indeed, in yeast, Cdc24 directly links the smw G protein Bud1 and Cdc42 by being a binding target for Bud 1 and a GEF for Cdc42 (8). In mammalian cells a putative Rac effector, Ost, is a GEF for Cdc42 and Rac (13). Also, RasGAP associates with p190RhoGAP, suggesting a possible interplay between these two G protein activators (18). Alternatively, GEFs or GAPs may be directly regulated by external stimuli resulting in altered activity or subcellular localization. For example, the activity of the Ras GEF Cdc25Mm is stimulated by muscarinic receptor-mediated phosphorylation in vivo (19). Also, the in vitro activity of the Rac GAP n-chimaerin is stimulated by phosphatidylserine and phosphatidic acid and inhibited by phospholipids such as lysophosphatidic acid (20).
Recently, our laboratory identified Graf, a GAP for
Rho associated with focal adhesion
kinase (FAK) following screening a chicken embryo gt11 expression
library with a radiolabeled carboxyl-terminal domain of FAK (21). This
protein contains a centrally located GAP domain followed by a
serine/proline-rich domain and a carboxyl-terminal SH3 domain. The Graf
SH3 domain was shown to specifically bind to a proline-rich region in
the COOH terminus of FAK. In vitro analysis revealed that
the Graf GAP domain enhanced GTP hydrolysis of Cdc42 and RhoA but not
Rac or Ras.
The experiments presented herein examine the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrate that Graf is ubiquitously expressed with high levels observed in heart and brain. Somewhat suprisingly, Graf phosphorylation is mediated by EGF/NGF signaling through MEK/MAP kinase. In vitro, purified MAP kinase catalyzes the phosphorylation of Graf on Ser510, a residue within the serine/proline-rich domain. The mutation of Ser510 to Ala inhibits EGF-induced phosphorylation of mutant Graf protein in vivo. Phosphorylation of Ser510 causes a slower mobility of Graf upon SDS-PAGE, suggestive of a conformational change in Graf. Together, these data indicate that Graf may be a site of convergence of growth factor signaling with Rho-mediated cytoskeletal changes.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture-- Cardiomyocytes from adult chicken ventricles were isolated by collagenase digestion using a Langendorf perfusion apparatus as described previously (22). Cells were lysed immediately for immunoprecipitation or plated on laminin-coated glass coverslips and maintained in serum free Dulbecco's modified Eagle's medium:F-12 (containing 1% penicillin/streptomycin) for immunocytochemistry. PC12 cells (obtained from ATCC) were maintained in RPMI containing 10% fetal calf serum, 5% horse serum, and a 1% penicillin/streptomycin solution. HEK 293 cells were maintained in Dulbecco's modified Eagle's medium:F-12 (1:1) containing 10% fetal calf serum and 1% penicillin/streptomycin.
Construction of Epitope-tagged Graf, Mutant Graf Protiens, GST Fusion Proteins, and Polyclonal Antibodies-- The cDNA construct encoding the NH2-terminal Flag-tagged variant of Graf (F-Graf; amino acid residues 1-584) was generated by PCR using primers that generated 5' HindIII and 3' XhoI restriction sites. The resultant PCR product was digested with HindIII and XhoI and ligated with HindIII and XhoI digested pcDNA3 DNA containing sequences encoding a 7 amino acid NH2-terminal epitope tag with the sequence, DYKDDDK. Mutations in Graf, Ser496 to Ala, Ser499 to Ala, and Ser510 to Ala were generated in F-Graf using PCR-based site-directed mutagenesis (Quick Change, Stratagene).
PCR-generated Graf cDNA sequences encoding amino acid residues 171-404 (GST-GrafGAP) and 471-584 (GST-GrafSH3) were cloned into pGex3X as described previously (21). FAK cDNA sequences that encode amino acid residues 686-1053 (CT and CT-P878A with a Pro to Ala mutation) were cloned into pGEX2TK as described previously (21). A fusion protein containing both the Graf GAP and SH3 domain (GST-GrafGAPSH, amino acid residues 171-584) was generated by PCR amplification using primers that generated 5' BclI and 3' XhoI restriction sites. The PCR product was digested with BclI and XhoI and ligated with pGex4T2 DNA digested with BamHI and XhoI. For antigen production, the GrafSH3 and GrafGAP fusion proteins were expressed in Escherichia coli and the fusion proteins purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech). The bound proteins were eluted with 20 mM glutathione (in 0.1 M Tris-HCl, pH 8.0, 0.12 M NaCl), and dialyzed against PBS (10 mM Na2HPO4, 0.15 M NaCl, 2.7 mM KCl, 1.2 mM KH2PO4). Rabbit polyclonal antiserum to GST-GrafGAP (Northern Blot Analysis-- RNA was isolated from chicken or rat tissues by phenol chloroform extraction using RNAzol (Tel-Test, Friendswood, TX), and mRNA was selected using oligo(dT)-Sepharose chromatography (Qiagen, Chatsworth, CA). RNA was resolved on a 1% agarose gel containing 2.2 M formaldehyde, transferred to nitrocellulose filters, and Northern blots containing chicken RNA were probed with a 1.5-kb Graf cDNA probe (nucleotides 285-1804). The mouse multiple tissue Northern blot (2 µg of poly(A+) RNA per lane, CLONTECH) and the rat Northern blot (containing 20 µg of total RNA) were probed with a 32P-labeled 0.9-kb mouse cDNA probe (corresponding to nucleotides 498-1400 of the chicken Graf sequence). The probes were labeled, hybridized, and the Northern blots washed as described previously (21, 24). Autoradiograms were obtained by exposing the blots to Kodak XAR film for 24-48 h.
Immunoprecipitation, in Vitro Association, and Western Blot
Analysis--
Tissue samples or cultured cells were lysed by
homogenization in a modified RIPA buffer (50 mM Hepes, 0.15 M NaCl, 2 mM EDTA, 0.1% Nonidet P-40 and
0.05% sodium deoxycholate, pH 7.2) containing 1 mM
Na3VO4, 40 mM NaF, 10 mM Na2 pyrophosphate. Graf was
immunoprecipitated by incubation of 1-2 mg of cell extract with the
polyclonal antibody, GGAP (5 µg/ml) for 2 h at 4 °C,
followed by a 1-h incubation with protein A-Sepharose-conjugated beads
(Pharmacia). The immune complexes were collected by centrifugation, the
beads were washed three times with RIPA buffer and once with TBS (0.2 M NaCl and 50 mM NaCl, pH 7.4), boiled in
sample buffer and the proteins resolved by 10% SDS-PAGE (25). Proteins
wre transferred to nitrocellulose, and a Western blot was performed
using the polyclonal antibody
GSH3 at a 1/500 dilution. Blots were
then incubated with HRP-conjugated Protein A (Amersham Pharmacia
Biotech) at a 1/1000 dilution, followed by visualization by
chemiluminescence (ECL, Amersham Pharmacia Biotech).
MAP Kinase Activity Assay--
MAP kinase was immunoprecipitated
by incubation of PC12 cell extract (100 µg) with the
p42MAPK-specific mAb (1B3B9; gift from Dr. Michael Weber)
as described above. The immune complexes were resuspended in MAP kinase
reaction buffer (25 mM Hepes, pH 7.5, 10 mM
magnesium acetate, and 50 µM unlabeled ATP) containing
myelin basic protein (2 µg) and [-32P]ATP (1 µCi)
and incubated at 30 °C for 20 min. The reaction mixtures were boiled
in SDS sample buffer, resolved by 15% SDS-PAGE, and visualized by
autoradiography.
In Vitro Phosphorylation of Graf--
Recombinant wild-type
p42MAPK was purified to apparent homogeneity as described
previously (26). Equal amounts of GST or GST-Graf fusion proteins
(approximately 2-6 µg) were incubated with purified MAP kinase (10 ng) and [-32P]ATP (1 µCi) in MAP kinase reaction
buffer. After incubation for 20 min at 30 °C, the reaction mixtures
were boiled in SDS sample buffer resolved by 14% SDS-PAGE and
visualized by autoradiography.
Phosphopeptide Mapping and Peptide Sequencing-- GST-Graf fusion proteins (phosphorylated by MAP kinase as described above) were digested with chymotrypsin as described previously (27). Phosphopeptides were spotted onto thin-layer chromatography (TLC) plates, resolved by high-voltage electrophoresis (40 min at 1,000 V in pH 8.9 buffer containing 0.295 M amonium bicarbonate), followed by chromatography in isobutyric acid buffer (27). Positions of phosphorylated peptides were determined by autoradiography. Individual phosphopeptides were isolated by aspiration onto a polyethylene disk (Omnifit) followed by elution in pH 1.9 buffer (27). 32P-Labeled phosphochymotryptic peptides were applied to Sequelon arylamine membrane (Milligen), dried, coupled, and washed as described previously (28). The membrane-bound peptide was placed into an Applied Biosystems 470A sequencer and cycled as described previously (29). The products of each cleavage reaction were collected, and 32P was detected by Cerenkov counting.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of Graf mRNA and Protein Expression-- Northern blot analysis using a chicken-Graf cDNA probe revealed a 5.0-kb Graf RNA in chicken brain and liver, consistent with previously published data (Fig. 1A; Ref. 21). In contrast, Northern analysis using a mouse Graf cDNA probe directed to the SH3 domain revealed a major 8.5-kb RNA species in most rodent tissues (Fig. 1B). The 8.5-kb transcript was most abundant in mouse heart and brain with lower levels expressed in kidney. Several smaller transcripts were observed in other mouse tissues, including a ubiquitous RNA species of approximately 4.5 kb and more restricted expression of RNAs of approximately 7, 2.5, and 2 kb. Similar results were observed when the blot was hybridized with a mouse Graf GAP domain cDNA probe (data not shown). As observed in Fig. 1, the major RNA species expressed in chicken co-migrates with a RNA expressed in rat (A) and may thus correspond to the 4.5-kb RNA species observed in the mouse Northern blot (B). Further evidence for alternative RNA processing arises from the identification of a variant of Graf (which contains sequences encoding a unique NH2-terminal domain) in a chicken brain cDNA library.2 A cDNA probe complementary to this alternative NH2-terminal sequence specifically hybridized with a transcript of approximately 2 kb, which was highly expressed in liver relative to brain, indicating that at least the 2-kb RNA species in liver may arise by alternative splicing. RNA from several cells lines was analyzed by Northern analysis, including RNA from REF52 (rat embryo fibroblasts), chicken embryo fibroblasts, Madin-Darby canine kidney epithelial cells, rat vascular smooth muscle cells, bovine endothelial cells, and PC12 rat pheochromocytoma cells. Of these, only PC12 cells expressed detectable levels of Graf RNA (data not shown). These results indicate that Graf transcription is broadly detectable in tissues with highest levels in brain and heart, but that its expression in tissue cultured cell lines is quite restricted.
|
|
Graf Expression and Phosphorylation in Cardiomyocytes and Neuronal Cells-- Since whole heart extracts contain high levels of Graf protein, we isolated cardiomyocytes to determine whether the expression in heart is due to the presence of Graf in these contractile cells. Both immunonocytochemistry and immunoprecipitation revealed the expression of Graf in isolated cardiomyocytes. Endogenous Graf in cardiomyocytes was localized in punctate structures along the actin stress fibers (data not shown), similar to the localization observed when Graf was ectopically expressed in chicken embryo fibroblasts (21). Western blot analysis revealed that Graf, immunoprecipitated from freshly isolated cardiomyocytes, exhibited a retarded mobility on SDS-PAGE compared with Graf immunoprecipitated from whole heart extracts (Fig. 3A). The mobility shift was reversed by treatment of Graf cardiomyocyte immunoprecipitates with calf intestinal alkaline phosphatase (CIP; Fig. 3B). These data indicate that Graf is phosphorylated in isolated cardiomyocytes and that phosphorylation appears to induce a change in mobility on SDS-PAGE.
|
|
|
Phosphorylation of Graf in Vitro and in Vivo on
Ser510--
The amino acid sequence of Graf predicts four
consensus sites for MAP kinase phosphorylation,
Pro-X-Ser/Thr-Pro, each located within the
serine/proline-rich region (Fig.
6A). To determine whether MAP
kinase catalyzed phosphorylation of Graf directly, a GST fusion protein
(GAPSH; Fig. 6A) containing the four phosphorylation consensus sites was incubated with purified MAP kinase and
[-32P]ATP. Fig. 6B shows that GAPSH was
efficiently phosphorylated by MAP kinase in vitro. A fusion
protein containing the three COOH-terminal consensus sites (SH3; Fig.
6A) was also phosphorylated by MAP kinase in
vitro. In contrast, neither GST alone nor a Graf fusion protein
containing the NH2-terminal most consensus site (GAP; Fig.
6A) was phosphorylated by MAP kinase.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have previously shown that Graf associates with FAK in vitro and has GAP activity for CdC42 and Rho (21). Since both integrin activation of FAK and signaling through the small molecular weight G proteins of the Rho family have been implicated in cellular adhesion, we considered the possibility that Graf may be involved in the convergence of these two pathways (21). In this report we have characterized the expression and possible regulation of the Graf protein. Our results show that Graf is highly expressed in brain and heart (and cells derived from these tissues), but not in fibroblasts or certain endothelial or epithelial cells. The tissue distribution of Graf is distinct from that of FAK, which is widely expressed in many tissues and cell lines. Notably, Graf is not found in cells wherein FAK has been shown to be important in adhesion-dependent signaling (e.g. fibroblasts); nor is Graf regulated (as assessed by tyrosine phosphorylation) by adhesion-dependent signaling or overexpression of FAK.2 In contrast, Graf phosphorylation appears to be regulated by EGF and NGF in PC12 pheochromocytoma cells, an event that correlates with the activation of MEK and MAP kinase.
The time course of Graf phosphorylation, inhibition of phosphorylation by PD098059, and the fact that MAP kinase phosphorylates Graf in vitro on sites that are also phosphorylated after EGF treatment in vivo all support the possibility that Graf is an in vivo substrate for MAP kinase in PC12 cells. The observation that Graf was phosphorylated in freshly isolated cardiomyocytes suggests that MAP kinase (or a related kinase) mediates this process. Interestingly, despite the fact that adult cardiomyocytes are terminally differentiated cells, these cells have been shown to contain significant levels of MAP kinase (34). In addition, increased coronary pressure can activate the MAP kinase cascade in these cells (34), an event that may occur during the cell isolation procedure upon reperfusion of the heart.
Although the data presented here are consistent with Graf being an in vivo target for MAP kinase, it is also possible that another MEK or MAP kinase activated kinase is responsible for phosphorylating Graf. MAP kinase phosphorylates and activates p90RSK as well as two recently identified Ser/Thr kinases, Mnk1 and Mnk2 (35-37). The time course of Graf phosphorylation and inhibition of phosphorylation by PD098059 would not rule out these candidate kinases. Notably Mnk2 is abundant in heart, but both Mnk1 and Mnk2 are expressed in very low levels in brain (36, 37).
We identified a major site of EGF-stimulated Graf phosphorylation as Ser510. Phosphorylation of this site correlates with a mobility shift of Graf on SDS-PAGE, suggestive of a conformational change in the protein. EGF may result in phosphorylation of a second site, since two slower migrating forms of Graf were detected in some experiments after EGF stimulation. The inability of the ectopically expressed S510A mutant to undergo a mobility shift after EGF treatment suggests that the second site of phosphorylation may be dependent on prior phosphorylation of Ser510. Interestingly, Ser510 resides in a proline-rich region adjacent to the carboxyl-terminal SH3 domain. An attractive possibility to explain the mobility shift is that the SH3 domain exhibits an intramolecular interaction with prolines in this region (e.g. closed form), and upon phosphorylation, the SH3 domain becomes conformationally altered (e.g. open form). Although there are no type I or II SH3 consensus binding sites in this proline-rich region (RXLPPXP and PPLPXR (boldface letters represent conserved residues), respectively), there are six PXXP motifs that form the core of the polyproline "type II" helix to which SH3 domains bind (38). Interestingly, the crystal structure of Src revealed that its linker region (which does not contain a consensus SH3 binding site) forms a polyproline helix that is sufficient for an intramolecular interaction with the Src SH3 domain (39). Thus, formation of a polyproline helix within the proline-rich region in Graf could direct an intramolecular interaction with the SH3 domain. Phosphorylation of Ser510 could relieve the intramolecular interaction and result in an open conformation with an exposed SH3 domain.
Given the above model for Graf regulation, one would predict that serine phosphorylation of Graf would enhance its binding efficiency for FAK. In the case of Sos1, serine phosphorylation has been shown to alter the affinity of Sos1 binding to the Grb2 SH3 binding domain (40). However, the low affinity interaction observed between FAK and Graf, and the lack of sensitivity of the in vitro association assays, have to date precluded us from thoroughly investigating this possibility. It is also possible that phosphorylation could modulate the ability of Graf to associate with another as yet unidentified SH3 binding partner. Graf has been shown to bind the FAK family member Pyk2, in vitro, via a PXXP motif; however, the low level of Pyk2 in heart suggests that this may not be a physiologically relevant binding partner.3
That a mitogenic signal may regulate Rho-mediated cytoskeletal changes is intriguing in light of the observation that MAP kinase itself has not been directly implicated in mediating cytoskeletal changes. In fact Ras-mediated membrane ruffling in fibroblasts and Ras-mediated cytoskeletal changes in cardiomyocytes do not require MAP kinase signaling (41, 42). However, MAP kinase is required for differentiation of PC12 cells into neuronal cells, a process that requires continued cytoskeletal changes (43, 44). EGF and NGF stimulate PC12 cell filopodial projections and membrane ruffling with a time course similar to that of MAP kinase activation (45, 46). Interestingly, inactivation of Rho by ADP-ribosylation with C3 endotoxin results in filopodial extensions in the neuroblastoma NEI-115 cell line (47). We are currently investigating whether EGF-mediated MAP kinase activation and subsequent phosphorylation of Graf could result in activation of Graf, down-regulation of Rho, and enhancement of neurite extensions.
The elevated levels of Graf in isolated cardiomyocytes are interesting
in light of recent evidence that implicates Rho signaling in cardiac
hypertrophy (48, 49). When embryonic cardiomyocytes are treated with
the 1-adrenergic agonist phenylephrine, cells undergo
drastic morphological changes that include complete reorganization of
the actin cytoskeleton in addition to various changes in gene expression. The changes in gene expression that accompany hypertrophy have been reported to require Rho signaling, since C3 can block the
phenylephrine-induced production of atrial natriuretic factor and the
contractile protein, myosin light chain-2 (49). We are currently
testing whether Graf can modulate this hypertrophic response in
vivo.
The physiological function of Graf is presently unknown. However, the
data presented above indicate that phosphorylation may play a role in
modulating Graf activity or interactions in vivo. Preliminary experiments indicate that phosphorylation of Graf does not
effect enzymatic activity, since microinjection of the Ser510 Ala mutant into Swiss 3T3 cells produces the
same dramatic cytoskeletal effects as wild-type Graf, an effect which
is dependent on GAP activity.2 Therefore, we hypothesize
that phosphorylation may regulate the ability of Graf to interact with
SH3 binding partners. Importantly, SH3 domain-mediating interactions
are often responsible for the intracellular targeting of proteins (38).
It will be of interest to determine whether the subcellular
localization of Graf is altered after mitogenic stimulation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Alan Richardson and Dr. Rajesh Malik for helpful suggestions. We thank Cheryl Borgman and Marlene Macklem for technical assistance and Dr. John Shannon for performing Edman degradation.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants CA29243 and CA40042 from the DHHS-NCI and Grant 4491 from the Council for Tobacco Research, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Research Service Award H1-F32- GM18297-01.
¶ Present address: Dept. of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
** To whom correspondence should be addressed: Dept. of Microbiology, Box 441, Health Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5395; Fax: 804-982-1071; E-mail: jtp{at}virginia.edu.
1 The abbreviations used are: smw, small molecular weight; MAP, mitogen-activated protein; MEK, MAP kinase kinase or Erk kinase; NGF, nerve growth factor; EGF, epidermal growth factor; GEF, guanine nucleotide exchange factors; GAP, GTPase-activating protein; SH3, Src homology 3 domain; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; TBS, tris-buffered saline; RIPA, radioimmunoprecipitation assay buffer; Ab, antibody; mAb, monoclonal antibody; p42MAPK, 42-kDa isoform of MAP kinase; kb, kilobase(s).
2 J. M. Taylor and J. T. Parsons, unpublished observation.
3 W. Xiong and J. T. Parsons, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|