©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Ligand for SH3 Domains
THE Nck ADAPTOR PROTEIN BINDS TO A SERINE/THREONINE KINASE VIA AN SH3 DOMAIN (*)

(Received for publication, December 22, 1994)

Margaret M. Chou (§) Hidesaburo Hanafusa (¶)

From the Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously shown that overexpression of the SH2- and SH3-containing Nck adaptor protein causes transformation of mammalian fibroblasts. To elucidate the mechanism by which it deregulates growth, we have sought to identify potential effectors for Nck. We report that a serine/threonine kinase, which we term NAK (for Nck-associated kinase), associates with Nck in vivo and in vitro. Using glutathione S-transferase fusion proteins generated with isolated domains of Nck, we demonstrate that NAK binds specifically to the second of Nck's three SH3 domains. NAK is complexed with Nck in a wide variety of cell types, including NIH3T3, A431, PC12, and HeLa cells.


INTRODUCTION

SH2 (^1)and SH3 domains are peptide motifs found in a wide variety of molecules that have demonstrated roles in regulating cell growth(1, 2) . It has been demonstrated that virtually all SH2-containing proteins bind to activated tyrosine kinases by a common mechanism(1, 2) . Specifically, engagement of growth factor receptors by their cognate ligands stimulates the intrinsic catalytic activity of the receptors, resulting in the autophosphorylation of multiple sites in their cytoplasmic domains(3) . These phosphorylation sites are recognized and bound by the SH2 domains of specific proteins. Binding of SH2-containing proteins to activated receptors can effect multiple responses. In most instances, the recruited protein acts as a substrate for the receptor. In the case of phospholipase C-, this tyrosine phosphorylation has been shown to enhance its specific activity(4) . For other molecules, however, no such modification of activity has been observed. Rather, it has been proposed that association with the receptor serves to juxtapose the protein with its physiological substrate. Such appears to be the case with the Ras GTPase-activating protein, whose target, p21, is localized at the plasma membrane(5) .

The effects of receptor binding have been more difficult to address for a distinct class of SH2-containing proteins, the so called adaptor proteins, which include p47(6) , the p85 regulatory subunit of phosphatidylinositol 3-kinase(7) , Nck(8) , Grb2/Sem-5(9, 10) , and Shc(11) . These adaptor proteins consist of little more than SH2 and SH3 domains and are believed to couple activated tyrosine kinases to various effector pathways. Since adaptor proteins possess no recognizable catalytic sequences, the molecular nature of their downstream effects has been more difficult to analyze. Despite this fact, understanding of the cellular pathways regulated by several adaptor proteins has grown rapidly in the last several years. For example, the p85 regulatory subunit of phosphatidylinositol 3-kinase serves to mediate interaction of the p110 catalytic subunit with activated growth factor receptors, resulting in its activation(7, 12, 13) . The second adaptor with a delineated effector is Grb2/Sem-5 (names of the murine and Cenorrhabditis elegans homologs, respectively)(9, 10) . Multiple investigators have recently shown that Grb2, in concert with Shc, functions in p21 activation by binding to the guanine nucleotide exchange factor for p21, mSOS(14, 15, 16) . Moreover, both biochemical and genetic evidence reveal that the SH3 domains of Grb2 are required for interaction with mSOS and for biological function(9, 10) .

The signaling pathways that are regulated by other adaptor proteins remain undefined. We have previously demonstrated that overexpression of Nck in mammalian fibroblasts results in transformation(17) . To better understand the precise role of Nck in regulating cell growth, we sought to identify molecules that interact with Nck. We and others (8, 17, 18, 19) have demonstrated that Nck binds to activated tyrosine kinases of both the receptor and cytoplasmic subtypes via its SH2 domain. However, little is known about the downstream effector pathways regulated by Nck. We previously described the association of Nck with a serine/threonine kinase in vitro(17) . In the current study, we demonstrate that Nck binds to a serine/threonine kinase in vivo and that this interaction is mediated by the second of Nck's three SH3 domains. Furthermore, they demonstrate that Nck binds to multiple kinases via distinct domains, potentially linking tyrosine kinases to a serine/threonine kinase pathway in the cell.


EXPERIMENTAL PROCEDURES

Cell Culture

3Y1 rat fibroblasts, all cell lines derived from 3Y1 (i.e. v-src transformed 3Y1 (SR3Y1), and nck-overexpressing Y1, Y2, and Y4 cell lines), and NIH3T3 murine fibroblasts were maintained in Dulbecco's minimal essential medium containing 5% bovine calf serum at 37 °C.

Plasmids and Constructs

Fusion proteins of various domains of Nck with glutathione S-transferase (GST) were generated. For the full-length Nck fusion protein (denoted GST-Nck), a polymerase chain reaction product flanked by BamHI linkers was inserted into the BamHI site of the pGEX-2T vector (Pharmacia Biotech Inc.). The fusion construct deleted in the first SH3 domain of Nck was obtained by subcloning the blunted, gel-purified AlwNI-XbaI fragment of nck into the blunted EcoRI site of the pGEX-3X plasmid (Pharmacia); this construct is denoted GST-332. For the fusion of GST with Nck lacking the first two SH3 motifs (GST-32), the GST-332 vector was partially digested with BstEII and BamHI, filled in, and religated. The chimeric protein consisting of GST with the SH2 domain of Nck (GST-2) was derived as follows. The GST-332 DNA was digested to completion with SmaI and StuI, and the 450-base pair fragment encoding the SH2 domain was gel purified and subcloned into the SmaI site of the pGEX-3X vector. Finally, the fusion protein encoding the second SH3 domain of Nck with GST (GST-3) was generated by digestion of the GST-332 plasmid with SpeI, followed by partial digestion with BstEII, filling in, and religation.

For the filter binding assays, we prepared an additional fusion protein construct in a pGEX-2T-derived plasmid, which was modified to encode a cAMP-dependent protein kinase phosphorylation site after the GST-encoding sequence(20) . The entire nck sequence, generated by polymerase chain reaction, was inserted into the BamHI site of this vector; this construct is denoted pGEX-2TK-Nck.

Preparation of Fusion Proteins

GST fusion proteins were prepared as previously described(17) . For the GST, GST-2, GST-32, and GST-332 constructs, isopropyl-1-thio-beta-D-galactopyranoside induction was carried out at 37 °C for 3 h; for the GST-Nck and GST-3 chimeras, induction was at 27-30 °C, as these proteins were trapped in inclusion bodies when induced at higher temperatures.

Preparation of Cell Lysates and Immunoprecipitation

Cells were washed twice with ice-cold phosphate-buffered saline and then lysed in NP-Tx buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 5 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 1% Trasylol, 1 mM phenylmethylsulfonyl fluoride). Alternatively, cells were lysed in RIPA buffer where indicated (10 mM Tris, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 10% glycerol, 0.1% SDS, 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, 1% Trasylol). Lysates were incubated on ice for 10 min and then pelleted in a microcentrifuge at 13,000 rpm for 10 min at 4 °C.

Immunoprecipitation, in Vitro Kinase Assays, and Phosphoamino Acid Analysis

For immunoprecipitations, anti-Nck (generated against GST-Nck fusion protein) antiserum was added to lysates and incubated for 1-2 h at 4 °C; 60 µl of a 1:3 slurry of protein A-Sepharose was added, and the lysates were incubated for 1 h more. For GST fusion protein precipitations, approximately 1-5 µg of fusion protein and 60 µl of glutathione-Sepharose beads (1:3 slurry) were used per precipitation. Immunoprecipitates/complexes were washed four times in NP-Tx buffer. For kinase assays, samples were then washed twice in kinase buffer (30 mM Tris pH 7.5, 10 mM MgCl(2), 1 mM dithiothreitol, 1 mM beta-glycerophosphate, 1 mM sodium vanadate, 1% Trasylol). Kinase reactions were carried out in a 30-µl volume of kinase buffer containing 5 µg of myelin basic protein (MBP) (Sigma) as exogenous substrate, 20 µM cold ATP, and 10 µCi of [-P]ATP (3000 Ci/mmol) (Amersham Corp.). Reactions were carried out at room temperature for 20 min and terminated by the addition of sample buffer, followed by boiling for 5 min. Samples were fractionated on 15% Laemmli gels and visualized by autoradiography. For assaying other substrates and conditions, 10 mM MnCl(2) was used rather than MgCl(2) where indicated, and casein, histone H1, or enolase was added instead of MBP as exogenous substrates.

Phosphoamino acid analysis was conducted as previously described(17) .

Substrate-containing Gels

Immunoprecipitations were carried out as described above and fractionated on 10% Laemmli gels containing MBP in the resolving phase at 0.5 mg/ml. The gel was treated as described (21) and equilibrated in kinase buffer (30 mM HEPES, pH 7.4, 10 mM MgCl(2), 10 mM MnCl(2), 2 mM dithiothreitol, 1 mM vanadate, 5 mM beta-glycerophosphate) for 30 min. Reactions were carried out for 1 h in 5 ml of the above buffer containing 100 µCi of [-P]ATP and 10 µM cold ATP. Finally, the gel was washed in copious amounts of 5% trichloroacetic acid, 1% sodium pyrophosphate for 10 h.

Filter Binding Assays

Gel electrophoresis and transfer of proteins to polyvinylidene fluoride membranes were carried out as previously described. Filters were blocked for 2 h in blocking solution (phosphate-buffered saline containing 2% bovine serum albumin and 0.1% Triton X-100). Filters were incubated with P-labeled GST-Nck, which was phosphorylated by cAMP-dependent protein kinase in vitro, as described by Kaelin et al.(20) . Blots were then washed four times for 15 min with phosphate-buffered saline, 0.1% Triton X-100 and subjected to autoradiography.


RESULTS

Nck Associates with a Serine/Threonine Kinase in Vivo

Work from several laboratories has shown that the Nck adaptor protein, which consists of three SH3 domains followed by one SH2 domain, associates with and is phosphorylated by multiple tyrosine kinases. However, virtually nothing is known about signaling downstream of Nck. We have previously demonstrated that Nck, expressed as a GST fusion protein, can bind to both serine/threonine and tyrosine kinases in vitro. Since the associated serine/threonine kinase represents a potential effector for Nck, we therefore sought to further characterize this interaction. To test whether this kinase associates with Nck in vivo, we immunoprecipitated Nck protein from 3Y1 rat fibroblast lysates. Immunocomplexes were washed and subjected to an in vitro kinase assay by incubation with [-P]ATP and MBP, a commonly used kinase substrate. Reaction mixtures were then fractionated by SDS-PAGE and visualized by autoradiography. These in vitro kinase assays revealed that Nck co-immunoprecipitates with a kinase that phosphorylates MBP (Fig. 1A). Phosphoamino acid analysis demonstrated that MBP in the anti-Nck immunoprecipitations was phosphorylated on both serine and threonine (Fig. 1B). In control pre-immune immunoprecipitations, a weak background kinase activity that phosphorylated MBP on serine only was detected. Thus, Nck appears to specifically associate with a serine/threonine kinase in vivo. We have termed this kinase NAK, for Nck-associated kinase.


Figure 1: Nck co-immunoprecipitates specifically with a kinase that phosphorylates MBP on threonine. A, in vitro kinase assay. Pre-immune (pi) or anti-Nck immunocomplexes were prepared from 250 µg of 3Y1 cell lysate as described under ``Experimental Procedures,'' washed, and subjected to an in vitro kinase assay by the addition of 10 µCi of [-P]ATP, 20 µM cold ATP, and 5 µg of MBP as exogenous substrate. Reactions were fractionated by 15% SDS-PAGE and visualized by autoradiography. Only the low molecular weight portion of the gel is shown, as MBP migrates in the 20-kDa range. B, phosphoamino acid analysis. The band representing MBP was excised and subjected to phosphoamino acid analysis as described. Lanes are labeled with the immunoprecipitating antiserum.



NAK Binds to the Second SH3 Domain of Nck

To determine which regions of the Nck protein are required for its association with NAK, various GST fusion proteins were generated with distinct domains of Nck (Fig. 2A). These fusion proteins were purified by adsorption to glutathione-Sepharose beads, which yielded essentially a single band by Coomassie staining (Fig. 2B). The purified fusion proteins were incubated with lysates of SR3Y1 cells, precipitated by the addition of glutathione-Sepharose, and subjected to in vitro MBP kinase assays. All of the GST fusion proteins coprecipitated a kinase(s) that phosphorylated not only MBP (Fig. 3A, bottompanel) but also the fusion protein itself (Fig. 3A, toppanel). Phosphoamino acid analysis of the MBP bands revealed that the SH2 domain of Nck alone was sufficient to precipitate a tyrosine kinase activity (Fig. 3B), which we have previously determined to be pp60(17) . (^2)More interestingly, however, a kinase that phosphorylated MBP on threonine was coprecipitated with the fusion constructs containing the second SH3 domain, namely the GST-Nck and GST-332. Deletion of the first SH3 motif had no effect on the association of NAK. Generation of a GST fusion protein with the second SH3 domain of Nck alone (GST-3) confirmed that this motif was sufficient for association of the threonine MBP kinase activity (Fig. 3A (rightpanel) and B). As a negative control, a GST chimera with the third SH3 domain of Nck was also tested; this construct failed to bind NAK (data not shown). A weak background kinase with no threonine kinase activity was also coprecipitated by all of the fusion proteins, including GST alone.


Figure 2: GST fusion constructs containing various domains of Nck. A, schematic diagram of GST fusion proteins. Portions of the Nck protein contained in each fusion protein are drawn, with SH3 domains stippled and SH2 darkstippled. The name of each construct is denoted at the right. The GST-Nck fusion protein represents the entire Nck molecule. B, Coomassie stain of purified GST fusion proteins. Bacteria expressing the various fusion proteins were lysed and incubated with glutathione-Sepharose beads. Proteins were eluted and dialyzed as previously described, fractionated by SDS-PAGE, and Coomassie stained. Fusion proteins are labeled at the top of each lane. The 27-kDa protein present in some of the samples (GST-3, GST-2, and GST-32) represents cleavage of the chimera to yield GST. Molecular masses in all figures are indicated in kDa.




Figure 3: The second SH3 domain of Nck mediates interaction with NAK. A, MBP kinase assay. Leftpanels, the indicated fusion proteins were incubated with SR3Y1 RIPA lysates, precipitated with glutathione-Sepharose, washed, and subjected to an in vitro kinase assay, SDS-PAGE, and autoradiography. The toppanel represents the top portion of the gel, in which the fusion proteins run; the bottompanel represents the low molecular weight portion of the gel where MBP runs. Amounts of fusion protein used vary (from 1-5 µg) as do exposure times for the various lanes. Rightpanel, GST-3 fusion protein was incubated with SR3Y1 lysate, precipitated, and subjected to a kinase assay. The MBP band is indicated with arrow; the upperband at 40 kDa represents the GST-3 protein. B, phosphoamino acid analysis. Phosphoamino acid content of the MBP bands are as in A, with the fusion protein used as the precipitating agent indicated at the top of each lane.



These results have several important implications. First, they identify a novel class of ligands for SH3 domains, namely serine/threonine kinases. Second, they confirm that Nck is an adaptor protein, binding to tyrosine kinases via its SH2 domain, potentially linking them to a serine/threonine kinase bound to its second SH3 domain. And third, they show that the SH3 domains of Nck are non-redundant, since neither the first nor third SH3 domains of Nck are necessary or sufficient for NAK association.

Substrate Specificity and Cation Requirements

To further characterize the Nck-associated kinase, we tested its ability to phosphorylate other substrates in vitro as well as its cation requirements. Anti-Nck immunocomplexes from 3Y1 cells were able to phosphorylate casein and histone but not enolase (Fig. 4). Phosphorylation of casein and histone occurred exclusively on serine (data not shown).


Figure 4: NAK substrates and cation requirements. Anti-Nck immunocomplexes were prepared from 3Y1 cells, split in five samples, and then subjected to kinase assays using various conditions. All lanes except lane2 utilize Mg in the reaction buffer. Lane1, MBP as exogenous substrate; lane2, MBP as substrate with Mn as cation; lane3, casein as substrate; lane4, histone H1 as substrate; lane5, enolase as substrate; lane6, kinase assay of anti-pp60 immunocomplex using enolase as substrate as positive control. Positions of enolase, H1, and casein are indicated with E, H, and C, respectively. Molecular masses are indicated in kDa.



Many serine/threonine kinases require the presence of Mg and are inactive when Mn is the only divalent cation present. However, we found that NAK functioned when Mn only was included in the kinase reactions (Fig. 4).

Substrate-containing Gels Reveal That Nck Binds to a Kinase of 65 kDa

To obtain a molecular size estimate of NAK, in-gel kinase assays were performed. As shown in Fig. 5, Nck co-immunoprecipitated with a kinase of approximately 65 kDa that phosphorylated MBP and that was not present in control immunoprecipitations. This protein was also detected by association with the GST-3 fusion protein but not GST alone. We assume that this kinase activity is directed toward threonine, since in all previous experiments this was the only amino acid specifically phosphorylated in anti-Nck immunoprecipitations. However, this could not be confirmed, as signals obtained by in-gel kinase assays were too weak to perform phosphoamino acid analysis.


Figure 5: Full-length Nck and its second SH3 domain coprecipitate with a kinase of 65 and 69 kDa in in-gel kinase assays. Whole cell lysates or immunoprecipitates were fractionated on Laemmli gels containing MBP (0.5 mg/ml) in the resolving phase, subjected to a kinase assay in situ, and visualized by autoradiography. WC, whole cell lysate (20 µg); lane1 is from 3Y1 cells, and lane2 is from the Y4 cell line. The remaining lanes are immunoprecipitates from 3Y1 cells and are labeled with the immunoprecipitating agent. Nck, anti-Nck immunoprecipitation; pi, pre-immune; GST, GST protein; and GST-SH3, fusion protein expressing the second SH3 domain of Nck alone. Arrow indicates migration of the 69-kDa kinase that coprecipitates with Nck, as well as its isolated SH3 domain.



Whole cell lysates of 3Y1 and a nck-overexpressing cell line (Y4) were also subjected to in-gel kinase assays but failed to reveal any differences in kinase activities between the two (Fig. 5, lanes1 and 2). This observation suggests that NAK activity may not be deregulated in nck-transformed cells. Such an interpretation is supported by experiments described below.

Interaction of Nck with 65- and 69-kDa Proteins by Filter Binding Analysis Is Conserved in a Wide Variety of Cell Types

Association of Nck with a 65- and 69-kDa protein could also be reproduced using filter binding assays. Lysates were prepared from various cell types, fractionated by SDS-PAGE, and transferred to filters. Following denaturation/renaturation, the filter was probed with a GST-Nck fusion protein labeled with P in vitro by phosphorylation by cAMP-dependent protein kinase. By this assay, Nck was found to bind to proteins of approximately 65 and 69 kDa in 3Y1 fibroblasts, as well as in nck- and v-src-transformed 3Y1 cells (Fig. 6). Neither of these proteins was observed to bind to GST alone (data not shown). Although the intensity of this 65- and 69-kDa doublet is greater in the Y2 nck-overexpressing cell line in this experiment, this variation is not reproducible and is dependent on the amount of lysate loaded. Immunoprecipitation of Nck from cells labeled with [S]methionine also showed a coprecipitating protein of approximately 69 kDa, indicating that association also occurred in solution (data not shown).


Figure 6: Nck binds to 65- and 69-kDa proteins by filter binding assays. Whole cell lysates (approximately 50 µg) were fractionated by SDS-PAGE and transferred to membranes. Membranes were then incubated with GST-2TK-Nck fusion protein, which was P labeled by in vitro phosphorylation with cAMP-dependent protein kinase. Filters were washed and visualized by autoradiography. Cell type is indicated at the top of each lane: A-E and A+E, A431 cells treated without or with EGF, respectively; ME16, 16-day-old whole murine embryos; 3T3-P and 3T3+P, NIH3T3 cells treated without or with platelet-derived growth factor, respectively. Arrows indicate 65- and 69-kDa proteins whose binding is conserved in all cell lines.



Interaction of Nck with this 65- and 69-kDa doublet of proteins was conserved in a number of other cell types, including human HeLa and A431 cells (Fig. 6). In A431 cells, binding occurred in an EGF-independent manner. Similarly, these proteins were detected in murine NIH3T3 fibroblasts as well as in lysates of 16-day-old murine whole embryos (Fig. 6). Again, binding of Nck to this doublet was growth factor-independent in NIH3T3 cells.

NAK Is Not Activated in nck- or v-src-transformed Cells

To ascertain what role NAK might have in mediating nck transformation, we examined the levels of NAK activity in nck-transformed cells. As shown in Fig. 7, NAK activity was unaltered in nck-overexpressing cell lines (Y1, Y2, and Y4) relative to parental 3Y1 cells. Experiments were performed to confirm that anti-Nck serum was not limiting (data not shown). NAK activity was similarly unaffected in SR3Y1 cells (Fig. 7).


Figure 7: NAK is not activated in nck- or v-src-transformed cells. Anti-Nck immunoprecipitates were prepared from 250 µg of lysate of various cell lines, indicated at the top of each lane, and subjected to in vitro MBP kinase assays. Y1, Y2, and Y4 are three nck-transformed cell lines, which overexpress low, high, and intermediate levels of nck, respectively.



NAK Localizes to P100 (Membrane) Fractions

Cell fractionation experiments were conducted to determine where NAK is localized within the cell. Furthermore, since no changes in total NAK activity were observed in the nck-transformed cell lines, we wished to explore the possibility that its subcellular location might be altered. Y1, Y2, and Y4 and parental 3Y1 cells were disrupted by hypotonic lysis and subjected to ultracentrifugation. Supernatants were collected as the soluble (S100) fraction and the resolubilized pellet as the membrane (P100) fraction. Purified GST-Nck fusion protein was added to both fractions, adsorbed to glutathione-Sepharose, and subjected to MBP kinase assays (Fig. 8A). Phosphoamino acid analysis revealed that NAK activity was only present in the P100 fraction in all cells (Fig. 8B). Appropriate fractionation was confirmed by probing for the presence of MAP kinase, known to be present in S100 (and nuclear) fractions(22) , by anti-MAP kinase immunoblotting (data not shown). NAK was also found to be exclusively in the P100 fraction in SR3Y1 cells (data not shown). Thus, membrane localization of NAK is not altered in nck- or v-src-transformed cells.


Figure 8: NAK localizes to P100 (membrane) fraction. A, the indicated cell lines were lysed hypotonically, subjected to ultracentrifugation, and the supernatant collected as S100 fraction (S); the pellet was resuspended and collected as P100 (P). GST-Nck fusion protein was added to each fraction, precipitated with glutathione-Sepharose, and subjected to a kinase assay. Migrations of GST-Nck and MBP are indicated. B, phosphoamino acid analysis of the MBP bands shown in A.




DISCUSSION

In this study, we demonstrate that Nck binds to a serine/threonine kinase via its second SH3 domain; we have termed this kinase NAK. These results have several important implications. First, they identify a novel class of ligand for SH3 domains, namely serine kinases. Second, they confirm that NCK acts as an adaptor protein, binding to tyrosine kinases via its SH2 domain, and potentially linking them to a serine/threonine kinase bound to its second SH3 domain. Third, they demonstrate that the SH3 domains of Nck are not redundant, since neither the first or third SH3 domains can bind NAK. To elucidate the mechanism by which nck transforms cells, we explored the role of NAK as a potential effector for Nck. Our data show that nck overexpression does not result in deregulation of NAK activity. This can be interpreted in several ways. One possibility is that the human Nck protein expressed in 3Y1 cells is incapable of interacting with the endogenous rat NAK. This seems unlikely, since incubation of these lysates with a GST-Nck fusion protein efficiently precipitates NAK activity in vitro (see Fig. 3). We favor the hypothesis that NAK exists at limiting levels in the cell, such that overexpression of Nck does not lead to increased coprecipitation of NAK activity. This idea is supported by immunodepletion experiments, where pre-clearance of NAK from 3Y1 lysates by immunoprecipitation of endogenous Nck vastly reduces the amount of NAK precipitable by exogenously added GST-Nck.^2 While this lack of increased NAK activity in nck-overexpressing cells may seem confounding, a similar result has been reported for the p85 regulatory subunit of phosphatidylinositol 3-kinase(7) . That is, phosphatidylinositol 3-kinase activity is not increased in cells overexpressing p85.

Our data further demonstrate that total NAK activity associated with Nck is not increased by a number of mitogenic stimuli, including v-src transformation, platelet-derived growth factor, EGF, insulin, nerve growth factor, and serum.^2 Thus, neither the NAK specific activity nor its association with Nck appears to be regulated by these stimuli. This is similar to what has been reported for Grb2 and mSOS: association of Grb2 with mSOS is independent of EGF stimulation. Moreover, purified Grb2 has no effect on the specific activity of mSOS(15) . It has been proposed that regulation of p21 occurs at the level of mSOS localization, such that ligand-induced autophosphorylation of the EGF receptor causes the recruitment of the pre-existing Grb2-mSOS complex to the membrane, where p21 resides. Regulation of a NAK substrate may also occur at the level of localization. It is tempting to speculate that NAK is not the sole effector of Nck but that other molecules associate with Nck's other SH3 domains and that the concerted actions of these proteins mediate nck transformation.

In the last several years, numerous groups have contributed to an increased understanding of how SH3 domains may function in cell signaling. Early studies (23) identified proline-rich regions as SH3 binding moieties. Furthermore, both direct biochemical and circumstantial evidence indicated that they play a role in G protein signaling. (i) The Grb2 adaptor protein binds to mSOS, an activator of the p21 protooncogene, primarily via its C-terminal SH3 domain(14, 15, 24) . (ii) Cicchetti et al.(25) have cloned an Abl-SH3 binding protein that possesses homology to GTPase-activating proteins for the Rho family of G proteins. (iii) A novel human Ras GTPase-activating protein specific for the CDC42 GTPase contains a proline-rich sequence that binds to the SH3 domains of c-Src and the p85 subunit of phosphatidylinositol 3-kinase. (iv) Dynamin, a neurally expressed G protein, binds to and is regulated by the SH3 domains of several signal-transducing molecules. Together, these observations suggest an interplay between SH3 motifs and G protein signaling.

More recently, other targets of SH3 domains have been identified. For example, phosphatidylinositol 3-kinase has been shown to interact with the SH3 domains of Src family kinases(26, 27) . Other reports indicate that the SH3 domain of Src also binds a serine/threonine kinase in vitro(28) , which appears to be distinct from NAK based on its substrate specificity. Interestingly, the SH3 domain of phospholipase C- is very homologous (46%) to the second SH3 domain of Nck, such that certain monoclonal antibodies against phospholipase C- cross-react with Nck(18) . Despite this fact, we do not observe a threonine-directed MBP kinase coprecipitating with intact phospholipase C- or with a GST fusion of its SH3 motif.^2 NAK therefore appears to be a specific effector for Nck.

Much work remains to elucidate the exact role of NAK in mediating Nck signaling. Identification of this kinase is a primary goal. Because the phosphorylation of MBP by NAK is on threonine, we explored the possibility that this might be a member of the MAP kinase/Erk family. However, we have excluded this based on immunoblotting of anti-Nck immunocomplexes with MAP kinase antibodies, and utilization of more specific substrates.^2 The results from the filter binding assays demonstrate that expression cloning may be a fruitful and expedient method for identifying Nck binding proteins. We are currently in the process of cloning such proteins. Isolation of these proteins will allow us to better understand how Nck regulates cell growth and how its overexpression mediates transformation.


FOOTNOTES

*
This work was supported by Grants R35CA44356 and T32CA09673 from NCI, National Institutes of Health, and VM2 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Cell Biology, Harvard Medical School, 25 Shattuck St., Boston, MA 02115.

To whom correspondence should be addressed: Laboratory of Molecular Oncology, Box 169, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8803; Fax: 212-327-7943.

(^1)
The abbreviations used are: SH2, Src homology 2; SH3, Src homology 3; SR3Y1, v-src-transformed 3Y1 cells; GST, glutathione S-transferase; MBP, myelin basic protein; NAK, Nck-associated kinase; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis.

(^2)
M. M. Chou and H. Hanafusa, unpublished observations.


ACKNOWLEDGEMENTS

We thank Gerd Blobel, Glen Scholz, and Beatrice Knudsen for critical reading of this manuscript.


REFERENCES

  1. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 [Medline] [Order article via Infotrieve]
  2. Koch, C. A., Anderson, D., Moran, M. F., Ellis. C., and Pawson, T. (1991) Science 252, 668-674 [Medline] [Order article via Infotrieve]
  3. Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212 [Medline] [Order article via Infotrieve]
  4. Nishibe, S., Wahl, M., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 248, 1253-1256
  5. McCormick, F. (1989) Cell 56, 5-8 [Medline] [Order article via Infotrieve]
  6. Mayer, B., Hamaguchi, M., and Hanafusa, H. (1988) Nature 332, 272-275 [CrossRef][Medline] [Order article via Infotrieve]
  7. Escobedo, J. A., Navankasattusas, S., Kavanaugh, W. M., Milfay, D., Fried, V. A., and Williams, L. T. (1991) Cell 65, 75-82 [Medline] [Order article via Infotrieve]
  8. Lehmann, J. M., Riethmuller, G., and Johnson, J. P. (1990) Nucleic Acids Res. 18, 1048 [Medline] [Order article via Infotrieve]
  9. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1992) Cell 70, 431-442 [Medline] [Order article via Infotrieve]
  10. Clark, S. G., Stern, M. J., and Horvitz, H. R. (1992) Nature 356, 340-344 [CrossRef][Medline] [Order article via Infotrieve]
  11. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Grignani, F., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93-104 [Medline] [Order article via Infotrieve]
  12. Skolnik, E. Y., Margolis, B., Mohammadi, M., Lowenstein, E., Fischer, R., Drepps, A., Ullrich, A., and Schlessinger, J. (1991) Cell 65, 83-90 [Medline] [Order article via Infotrieve]
  13. Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell 65, 91-104 [Medline] [Order article via Infotrieve]
  14. Buday, L., and Downward, J. (1993) Cell 73, 611-620 [Medline] [Order article via Infotrieve]
  15. Gale, N. W., Kaplan, S., Lowenstein, E. J., Schlessinger, J., and Bar-Sagi, D. (1993) Nature 363, 88-92 [CrossRef][Medline] [Order article via Infotrieve]
  16. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T. (1992) Nature 360, 689-692 [CrossRef][Medline] [Order article via Infotrieve]
  17. Chou, M. M., Fajardo, J. E., and Hanafusa, H. (1992) Mol. Cell. Biol. 12, 5834-5842 [Abstract]
  18. Meisenhelder, J., and Hunter, T. (1992) Mol. Cell. Biol. 12, 5843-5856 [Abstract]
  19. Park, D., and Rhee, S. G. (1992) Mol. Cell. Biol. 12, 5816-5823 [Abstract]
  20. Kaelin, W. G., Krek, W., Sellers, R., DeCaprio, J. A., Ajchenbaum, F., Fuchs, C. S., Chittenden, T., Li, Y., Farnham, J., Blanar, M. A., Livingston, D. M., and Flemington, E. K. (1992) Cell 70, 351-364 [Medline] [Order article via Infotrieve]
  21. Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143 [Medline] [Order article via Infotrieve]
  22. Chen, R., Sarnecki, C., and Blenis, J. (1992) Mol. Cell. Biol. 12, 915-927 [Abstract]
  23. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993) Science 259, 1157-1161 [Medline] [Order article via Infotrieve]
  24. Simon, M. A., Dodson, G. S., and Rubin, G. M. (1993) Cell 73, 169-177 [Medline] [Order article via Infotrieve]
  25. Cicchetti, P., Mayer, B. J., Thiel, G., and Baltimore, D. (1992) Science 257, 803-806 [Medline] [Order article via Infotrieve]
  26. Pleiman, C. M., Clark, M. R., Timson Gauen, T. K., Winitz, S., Coggeshall, K. M., Johnson, G. L., Shaw, A. S., and Canbier, J. C. (1993) Mol. Cell. Biol. 13, 5877-5887 [Abstract]
  27. Liu, X., Marengere, L. E. C., Koch, C. A., and Pawson, T. (1993) Mol. Cell. Biol. 13, 5225-5232 [Abstract]
  28. Weng, Z., Taylor, J. A., Turner, C. E., Brugge, J. S., and Seidel-Dugan, C. (1993) J. Biol. Chem. 268, 14956-14963 [Abstract/Free Full Text]

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