Identification of a human epidermal growth factor receptor-associated protein kinase as a new member of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase family.

A putative mitogen-activated protein kinase (MAPK) has recently been identified, which potentially phosphorylates the human epidermal growth factor (EGF) receptor at a physiological site (Thr-669) and is distinguished from other MAPKs/extracellular signal-regulated protein kinases (ERKs) on the basis of chromatographic, immunological, and kinetic data. Here we report that this newly discovered MAPK is physically associated with the EGF receptor in A431 cells and with the related receptor/tyrosine kinase HER2 (encoded by c-neu) in enzyme preparations obtained from Wilm's tumors. This human EGF receptor-associated kinase is characterized as a 40-kDa Thr-669 kinase that exists in a high molecular mass complex with the respective growth factor receptor. EGF treatment of A431 cells stimulates the tyrosine phosphorylation of p40 and increases Thr-669 kinase activity in p40-containing fractions. The 40-kDa kinase is recognized by affinity-purified polyclonal antibodies directed against the sea star p44mpk and a Pan-ERK antibody directed against the conserved subdomain VIII of MAPKs/ERKs, but is not recognized by antibodies selective for the rat p44erk1 and/or the p42mapk/erk2 isoforms, thus identifying the EGF receptor-associated kinase as a novel MAPK that may regulate receptor function in vivo.

* This research was supported in part by grants from the National W.), and the National Cancer Institute of Canada (to S . L. P.) and by contributions from Vernon To10 and the John C. Wilson, Jr. Endowment (to F. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor; Mops, 4-morpholinepropanesulfonic acid; MBP, myelin basic protein; WGA, wheat germ agglutinin; Ab, antibody; mAb, monoclonal antibody; PDPK, proline-directed protein kinase. nases implicated in a diversity of cell signaling pathways (1)(2)(3)(4).
Often stimulated directly by phosphorylation on both tyrosyl and serylkhreonyl residues (5)(6)(7)(8)(9)(10), MAPKs may function as convergence points in integrated protein kinase cascades that mediate cellular activation (11)(12)(13). Several serindthreonine kinases that represent distal targets in growth factor action, such as ~7 4 '~~ (14,151 and the ribosomal S6 kinases ~8 5 ' "~ (16, 17) and ~7 0~~~ (18), are directly phosphorylated in vitro by M A P % at physiological sites. However, W K -r e l a t e d enzymes have also been implicated in the regulation of more proximal aspects of molecular signaling by governing the biological activity of growth factor receptors. The human EGFclass of receptors in particular are known to be phosphorylated at Thr-669 in vivo (19, 201, a site that is phosphorylated by a number of MAPK isoforms in vitro (21)(22)(23). Recently, we identified a novel putative MAPK, which potentially phosphorylates the human EGF receptor Thr-669 site and is distinguished from other MAPKdERKs on the basis of chromatographic, immunological, and kinetic data (23). In the present study we report that this newly discovered MAPK is physically associated with the EGF receptor in humanA431 cells and with the related receptorltyrosine kinase HER2 (encoded by c-neu) in enzyme preparations obtained from Wilm's tumors. We describe the physiological activation, tyrosine phosphorylation, and partial purification of the enzyme. In addition, we characterize a profile of immunoreactivity that identifies this human EGF receptor-associated kinase as a MAPK but distinguishes this enzyme from previously identified members of the mammalian MAPWERK family.

Materials
Cells and Tissues-Human A431 epidermoid carcinoma cells obtained from the American Type Culture Collection (ATCC CRL 1555) and maintained as monolayers in log phase growth (25) were incubated in the absence of 10% fetal calf serum for 3 h prior to stimulation with 5 ng/ml murine epidermal growth factor (EGF) and harvesting as described by Sanghera et al. (23). Human pediatric Wilm's tumors provided by Timothy Triche (Chief of Pathology) and Phyllis Pepe of Children's Hospital Los Angeles were flash-frozen in liquid nitrogen and stored frozen at -70 "C prior to homogenization and enzyme purification.
Other Reagents-Wheat germ agglutinin-Sepharose, chromatographic media, and HR 515 Mono Q columns were purchased from Pharmacia LKB Biotechnology Inc. Murine EGF was purchased from Collaborative Research.

Methods
FPLC Mono Q Chromatography-Human A431 cell extracts were prepared as described in Sanghera et al. (23). Approximately 4 mg of clarified, filtered detergent lysate was applied to an equilibrated HR 515 Mono Q column in Buffer A (5 m~ Mops, pH 7.2, 5 m M EGTA, 1 m M sodium vanadate, and 1 mM dithiothreitol) a t a flow rate of 0.8 mVmin on a Pharmacia FPLC system. The column was developed with a 10-ml linear 0-800 mM NaCl gradient in Buffer A mixed and delivered a t the same flow rate; 0.25-ml fractions were collected. Selected column fractions were immediately analyzed by in vitro kinase assays or were preserved with SDS sample buffer for Western analysis.
Protein Kinase Assays and Western Analysis-Chromatographic fractions were assayed for their phosphotransferase activity toward 1 m g / d myelin basic protein or 1 m M EGF receptor Thr-669 peptide substrate (ELVEPLTPSGEAPNQALLRKK), as described previously (23,401. Mono Q fractions were subjected to SDS-PAGE, electrophoretic transfer to Immobilon P membranes (Millipore), and immunoblotting as described (23)(24)(25)32). Amnity-purified, alkaline phosphatase-or horseradish peroxidase-conjugated secondary antibodies were generally used a t a 1:lOOO dilution in 1% bovine serum albumin. Enzymatic detection was routinely performed with 5-bromo-4-chloro-3-indolyl phosphatel nitro blue tetrazolium or 3.3"diaminobenzidine tetrahydrochloride respectively; however, an enhanced chemiluminescence (ECL) detection system (Amersham Corp.) was also utilized, as noted in legend to Fig. 4. Specific immunoprecipitations and affinity precipitations with wheat germ agglutinin-Sepharose were performed as described in the legend to Fig. 4.
Partial Purification of HERA Kinase from Wilm's "Lmors-One hundred grams of solid Wilm's tumor were homogenized and sonicated in a protective buffer (32), clarified by centrifugation, and applied to a 100-ml quaternary amine-Sepharose (Pharmacia Fast Q) column, which was washed extensively with 350 m M NaCl in equilibration buffer (as described in Ref. 32, plus vanadate) to elute the identified MAF'Ks (4,6,12,23), followed by step elution with 700 m M NaCl, dilution to 350 mM NaCl, and application to a second Fast Q column (25 ml), which was washed and developed with a linear 350-800 m M NaCl gradient. The respective peak of Wilm's tumor Thr-669 peptide kinase activity eluted from this column at approximately 500 m M NaCl. Concentrated aliquots were stored at -70 "C.

RESULTS AND DISCUSSION
FPLC Mono Q fractionation of soluble extracts from human A431 epithelioid carcinoma cells previously afforded the resolution of four distinct peaks of EGF receptor Thr-669 phosphotransferase activity, three of which (peaks 11,111, and IV) were stimulated by treatment of A431 cells with EGF after a 3-h period of serum deprivation (23). These EGF-activated Thr-669 kinase peaks 11, 111, and IV were also readily detected with myelin basic protein (MBP) used as an alternative substrate ( Fig. 1, a and b). The Thr-669hIBP phosphotransferase activity in Mono Q peak I1 was previously attributed to p42mapk (ERK2) and a 44-kDa MAPK that appears to be encoded by the erkl gene (23). The Mono Q peak I11 kinase did not cross-react with any of five distinct MAPK antibodies at our disposal; however, Western analysis of peak I11 with monospecific antibodies detected a subset of A a nd ~3 4 "~"~, the regulatory and "X FRACMN NUMBER

FIG. 1. FPLC anion exchange chromatography resolves three
putative EGF receptor Thr-669 kinase activities in human A431

cells.
A Triton X-lOO/Nonidet P-40-solubilized extract (4 mg of protein prepared as in Ref. 23) from untreated cells (0, A) or from cells treated for 5 min with 5 ng/ml EGF (0, A) was applied to an equilibrated HR 515 Mono Q column followed by elution of the bound protein with a steep linear NaCl gradient, as described under "Experimental Procedures." Panels a and b, the column fractions (0.25 ml) were assayed for phosphotransferase activity toward 1 mg/ml MBP (0,O) (Sigma) and 1 m M Thr-669 peptide (A, A). The location of three distinct peaks of EGF receptor Thr-669/MBP kinase activity (11,111,and IV) identified in previous studies (23) under identical conditions are as indicated. Aliquots (75 pl) of selected Mono Q fractions obtained from EGF-stimulated cells were subjected to Western blotting procedures with the following probes: panel c, a commercial antibody that recognizes the human EGF receptor (Cambridge Research Biochemicals, Oncoprotein Antibody 0A-11-852); panel d, affinity-purified antibodies directed against purified sea star p44'"pk (24); panel e, affinity-purified antibodies directed against the carboxyl-terminal 34 residues of mouse cdc2 kinase (anti-Cdc2-CT). The identity of the immunoreactive protein in panel c as the EGF receptor was confirmed by the comigration of partially purified EGF receptor (membrane preparations) obtained from A431 cells, and the affinity of this protein for wheat germ agglutinin. Authentic ~3 4~~~ detected with anti-Cdc2-CT Ab migrates just below the position of the prestained standard carbonic anhydrase (33 kDa) (see Ref. 23), which marks the boundary ofpanel e. Similar results were obtained in three separate experiments. catalytic subunits, respectively, of the proline-directed protein kinase (PDPK) (25). The EGF stimulation of Mono Q peak I11 is remarkable in that p34CdC2/p58CYc1in A PDPK was initially identified in mammalian cells as a growth factor-sensitive enzyme system (26,27), a finding that has been confirmed in a number of different cells (28), as well as platelets (29). Mono Q peak IV contains a novel member of the MAP kinase family that, in addition to chromatographic behavior, can be distinguished from other putative EGF receptor Thr-669 kinases on the basis of immunoreactivity and enzyme kinetics (23). This Mono Q peak IV kinase was tentatively identified as a 40-kDa protein that cross-reacts with a polyclonal antibody directed against the purified sea star MAP kinase ( a n t i -~4 4~p~ Ab) (see Fig. Id but not by any of a series of antibodies directed against defined epitopes within other identified MAPWERKs (23, 24). Subsequent studies were undertaken to better characterize the Mono Q peak IV kinase and its physical association with human EGF-class receptors.
The observed cross-reactivity of the 40-kDa protein in Mono Q peak IV with the a n t i -~4 4~p~ antibody involves epitopes other than phosphotyrosine, since the presence of 1 m~ phosphotyrosine was demonstrated to have no effect on the resulting immunoreactivity (not shown). Remarkably, the putative 40-kDa protein kinase in Mono Q peak IV did not cross-react with several monospecific antibodies selective for the identified mammalian MAPWERKs. However, this 40-kDa protein was recognized by an affinity purified antibody (Upstate Biotechnologies Inc., anti-Cdc2-CT) generated against the carboxylterminal 34 residues of the murine cyclin-dependent protein kinase ~3 4 "~'~ (Fig. le), a sequence that spans kinase subdomain XI (30). To a lesser extent, p42mapk, but not ~44"'~l, in Mono Q kinase peak I1 was also detected with this a n t i -~3 4 "~=~ antibody. The consistent and specific cross-reactivity of the novel 40-kDa MAPK with anti-Cdc2-CT, but not with a second antibody raised against the seven most carboxyl-terminal residues of murinehuman ~3 4 '~'~ (DNQIKKM), demonstrated that the epitopes exhibiting cross-reactivity can be mapped to ~3 4 "~"~ subdomain XI.
The comparative immunoreactivities of p42mapk, ~44"'~', and the novel 40-kDa putative MAPK were further examined in side-by-side Western blots of Mono Q peak I1 fractions versus Mono Q peak IV fractions from non-stimulated cells. As shown in Fig. 2, both p42mapk and ~4 4 " '~' were readily detected with the mpk-I Ab (24), anti-ERK-I Ab, and a n t i -~4 4~p~, antibodies, whereas the 40-kDa protein in Mono Q peak IV reacted only with the latter of these three antibodies (also see Ref. 23). To further establish that p40 is a novel member of the MAPWERK family, we developed an additional Pan-ERK antibody directed against the highly conserved subdomain VIII, which includes the dual (TEY) regulatory phosphorylation sites (1). In addition to recognizing the p42""Pk and ~4 4~'~~ proteins in Mono Q peak 11, appreciable immunoreactivity toward the 40-kDa putative MAPK in Mono Q peak IV was observed with this Pan-ERK antibody, which is interesting in that this anti-peptide antibody (generated against a dephosphopeptide) would be expected to recognize only the inactive, nonphosphorylated species of these enzymes. The observed immunoreactivity of p40 with the Pan-ERK antibody is substantially less than that of the p42mapk and ~4 4 " '~' proteins, which suggests that the primary structure of p40 is either somewhat divergent or that one or both of the (: EX) regulatory sites may be phosphorylated. In these comparative Western analyses, strong immunoreactivity of the novel p40 MAPK with anti-Cdc2-CT antibodies was consis- Mono Q peak IV is characterized as a -40-kDa protein that is recognized by antibodies directed against 1) the purified sea star p44"pk (anti-p44"pk Ab), 2) subdomain XI of murine p34cdc2 (anti-Cdc2-CT), and 3) the conserved sequences within MAP/ERK subdomain VI11 (anti-Pan-ERK antisera) to a lesser extent. The anti-Pan-ERK blot was overdeveloped to reveal the comparatively weak but consistent immunoreactivity of p40; recombinant GST-ERK 1 expressed in Escherichia coli is used here as a positive control.
tently observed, whereas the immunoreactivity of p4ZrnaPk was comparatively weak and cross-reactivity with ~4 4 " '~~ was not observed. The detection of p40 in Mono Q Peak IV with anti-p44mpk and anti-Cdc2-CT antibodies, as well as Pan-ERK antibodies, in the absence of EGF pretreatment (compare with Fig. 1) further suggests that enzyme activation may not be a prerequisite for EGF receptor binding (also see below).
The immunoreactivity of the putative 40-kDa MAP kinase identified in Mono Q peak IV with an antibody based upon ~3 4 "~"~ sequences is not surprising, since MAP kinases are highly related to ~3 4 "~"~ in primary structure (1). Furthermore, kinase subdomain XI, which is encompassed by the peptide used to generate the anti-Cdc2-CT antibody, is a region of particularly high amino acid identity. Within this region, p42mapWerk2 is somewhat more closely related to murine ~3 4 "~"~ than is ~44"'~', which may account for the appreciable difference in immunoreactivity of p42mapWerk2 versus ~4 4~'~' in Mono Q peak I1 observed with this anti-Cdc2-CT antibody (see Fig. le). The strong cross-reactivity of the 40-kDa putative MAPK in Mono Q peak IV with antibodies recognizing two conserved protein kinase subdomains (subdomains VI11 and X I ) is meaningful in that this finding provides additional confirmation that p40 is a protein kinase. Perhaps significantly, of all the known MAP kinases, p4OfUs3 from Saccharomyces cerevisiae displays the greatest amino acid sequence identity to murine ~3 4 "~"~ in kinase subdomain XI (31). Furthermore, p4OfUs3 is the least conserved of the known MAP kinases to rat ~4 4 "~~~ at the carboxyl terminus. Accordingly, antibodies directed against the carboxyl-terminal 35 residues of rat ~4 4 " '~~ (anti-erkl-CT) immunoblotted both p42mapk and ~44"'~' in Mono Q peak 11, but not the 40-kDa protein in Mono Q peak IV (23). Taken together with the biochemical data, these immunochemical findings indicate that the 40-kDa protein in Mono Q peak IV is a protein kinase. It appears to be a MAP kinase, perhaps most closely related to the yeast p4OfUs3 based upon its apparent molecular size and profile of immunoreactivity.
To test the hypothesis that one or more of these putative EGF receptor Thr-669 kinases identified in human A431 epithelioid carcinoma cells may be physically associated with this growth factor receptor (231, we performed Western analysis of each of the respective FPLC Mono Q peak fractions. As shown in Fig. IC, the majority of EGF receptor immunoreactivity co-eluted with Mono Q peak 11, although a substantial portion of this receptor also co-eluted with Thr-669 kinase activity in peak IV. Although an interaction between PDPK and the EGF receptor would be in accord with the concept that the multifunctional PDPK may serve a triggering function at the mammalian R point (i.e. during the G1 to S phase transition) (28, 321, only traces of the receptor were detected in those fractions corresponding to peak 111. FPLC Superose 6 gel filtration analysis of Mono Q peak I1 revealed quantitative recovery of MBP kinase activity at an apparent molecular mass of approximately 44,000, which precludes a physical association between the EGF receptor and either p42mapWerk2 or ~4 4 " '~~ under these conditions. By contrast, the major MBP kinase in Mono Q peak IV displayed an apparent molecular mass of approximately 420,000 upon gel filtration, which taken together with the detection of a 40-kDa anti-p44"pk reactive protein by SDS-PAGE and Western blotting of the high molecular weight fractions (23) indicates that this kinase does indeed reside in a multiprotein complex. This conclusion is further supported by the finding that most of the MBP kinase activity in the Mono Q peak IV fractions can be adsorbed by wheat germ agglutinin (WGA)-agarose (data not shown).
In an effort to isolate a larger amount of this Mono Q peak IV kinase and to further characterize the relationship with the EGF-class of receptors, we undertook to purify this enzyme from Wilm's tumors, utilizing preparative (Pharmacia Fast Q ) rather than analytical (FPLC Mono Q ) anion exchange chromatography followed by FPLC Superose 12 gel permeation chromatography. After eliminating the identified M A P & by a series of ion exchange steps (see "Experimental Procedures"), the respective Wilm's tumor Thr-669 peptide kinase was eluted by a high salt gradient (350-800 m M NaC1) and an aliquot of the peak kinase activity was applied to a calibrated Superose 12 sizing column (Fig. 3a). The resulting Thr-669 kinase activity was determined to reside in a high molecular mass complex with an apparent molecular mass greater than 400 kDa. The peak activity fractions contained several proteins in the 120-200-kDa range that were phosphorylated in vitro upon the addition of [Y-~~PIATP (Fig. 3b). Western blotting of Superose 12 fractions, as well as peak anion exchange column fractions, revealed traces of EGF receptor; however, HER2 was readily identified as a prominent protein that co-fractionated with both Thr-669 kinase activity and a n t i -~4 4~p~ Ab immunoreactivity (Fig. 3, b and c). This is an interesting finding in that fetal kidney is exceptionally rich in HER2 (33). As in A431 cells (23), the 40-kDa protein from Wilm's tumors did not cross-react with the anti-mpk-I Ab (Fig. 3c), which readily detects p42rnapWerk2 and ~4 4~'~' .
These results indicate that a similar if not identical MAPK is present in a high molecular weight complex with the EGF receptor in A431 cells and with HER2 in Wilm's tu- fractionation of HER2 with the major peak of Thr-669 kinase activity is demonstrated by SDS-PAGE and Western blotting of peak fractions with a monoclonal antibody (Oncogene Science, Ab-2) specific for c-neul HER2 (36). The identity of the immunoreactive doublet as HER2 protein was confirmed by including control ( c ) membrane preparations obtained from HER2-transfected NIH 3T3 cells, which overexpress this protein (27). The accompanying SDS-PAGE gel is stained with Coomassie Blue, while the accompanying autoradiograph identifying high molecular weight phosphoproteins was obtained upon incubating a 25-pl aliquot of the indicated fraction for 20 min at 30 "C in the presence of [y-32P]ATP, followed by SDS-PAGE. Panel c, Western analysis of Superose 12 peak Thr-669 kinase fractions with affinity purified polyclonal antibodies directed against the purified sea star p44"' pk (anti-p44"' Pk) readily recognize the 40-kDa MAPK isoform present in these high molecular weight complexes (arrow). However, rabbit anti-peptide antibodies generated against the p44"pk sequence GLAYIGEGAYG-MVC (anti-mpk-l), which recognize p44'"pk, p42mapWerk2, and ~4 4~'~' isoforms on Western blots (23, 24), failed to detect this protein. In this experiment, a MAF'K preparation that reacts with both antibodies was utilized as a positive control ( c ) in the development of the Western blots.
mors. Based on this finding, as well as the ability to phosphorylate the EGF receptor Thr-669 phosphorylation site, this novel 40-kDa protein kinase is operationally designated Euman EGF Receptor-Associated Kinase (p40HERAK), until microsequence information and/ormolecular cloning can place this enzyme more specifically within a subfamily of the prolinedirected protein kinases (1,18,28). Precedent for the observed physical association between a growth factor receptorltyrosine kinase and a distinct serindthreonine protein kinase has recently been established in the case of the PDGF receptor and the cytoplasmic oncogenic kinase p74' *"l (34).
The utilization of large scale Wilm's tumor preparations enabled additional FPLC sizing runs, providing sufficient material to visualize the p40HERAK upon Coomassie Blue staining of SDS-PAGE gels (Fig. 4u). The identity of this protein as the novel MAPK was confirmed by subsequent immunoblotting of peak fractions. The Wilm's tumor preparations also enabled the assessment of additional affinity interactions with receptor proteins. As shown in Fig. 4b, the p40HERAK was absorbed by wheat germ agglutinin-Sepharose, which is generally selective for membrane glycoproteins. Moreover, the p40HERAK was detected in HER2 immunoprecipitates only in the presence of the primary antibody (Fig. 4c). These results confirm the physical association, if not the physiological relationship, between p40HERAK and HER2, providing additional routes for future purifications, subsequent microsequencing, and eventual cloning of this potentially important enzyme.
The growth factor sensitivity of p40HERAK observed in human A431 cells was further investigated by comparative immunoblotting. As shown in Fig. 5a, the levels of p4O immunoreactivity detected with anti-p44"pk Ab were not significantly altered by EGF pretreatment, which suggests that the levels of this protein in Mono Q peak TV do not depend on prior receptor sponding to the high molecular weight peak of Thr-669 kinase activity obtained from FPLC Superose 12 chromatography of Wilm's tumor preparations (see Fig. 3) were subjected to SDS-PAGE on a 6 1 5 % gradient gel and stained with Coomassie Blue. The identity of the 40-kDa protein, which was present in this high molecular weight complex (arrow) and which correlated precisely with Thr-669 phosphotransferase activity (see Fig. 3a), as p40HERAK was confirmed by align-Panel b, absorption of p40HERAK to WGA was confirmed by incubating ment of bands following Western analysis with anti-p44'"pk antibodies. 120 pl of the Wilm's tumor Fast Q peak IV fractions with 30 pl of WGA-Sepharose (Pharmacia) on ice for 5 min followed by two washes with Fast Q buffer, addition of 2 x SDS buffer to the resulting pellet, SDS-PAGE, and Western blotting. The detection of p40mRAK (arrow) in the resulting precipitates was confirmed by immunoreactivity with anti-p44'"pk antibodies, utilizing horseradish peroxidase-labeled secondthe physical association of p40HERAK with the HER2 was confirmed by ary antibodies and 3,3'-diaminobenzidine tetrahydrochloride. Panel c, incubating 100-pl aliquots of the Fast Q peak fractions in the presence (+) or absence (-) of a monoclonal antibody specific for c-neuEIER2 (Oncogene Science, Ab-2), followed by precipitation with protein G-agarose, washing two times with Fast Q buffer, and Western analysis with Amersham) detection system. anti-p44"Pk antibodies utilizing an enhanced chemiluminescence (ECL, phosphotyrosine content. Following a 3-h period of serum deprivation, human A431 cells were treated with or without 5 ng/ml EGF for 5 min, as previously described (23) with the exception that the subsequent lysates were immediately flash-frozen in dry icdmethanol prior to storage a t -70 "C. (Note: this procedural modification resulted in the preservation of a greater -fold stimulation of p42mapWe'k2/p44erk' (Mono Q peak 11) activity.) FPLC Mono Q chromatography was performed on each sample, and fractions corresponding to peak IV were collected and analyzed by Western blotting with a n t i -~4 4~p " Ab and PY20 anti-phosphotyrosine mAb, respectively. The lane numbers correspond to the Mono Q fractions that bracket peak IV kinase activity. As shown in a, the levels of p40mRAK immunoreactivity detected by the anti-p44"pkAb were not affected by the presence or absence of EGF. However, PY20 anti-phosphotyrosine mAb immunoreactivity ( b ) was detected in a band that co-migrates precisely with p40mRAK only in extracts of EGFtreated cells. Superimposition of the two blots confirmed that the band reactive with anti-p44"pk Ab exactly coincides with a band of antiphosphotyrosine immunoreactivity.
activation. In contrast, Western blotting with anti-phosphotyrosine antibodies detected phosphotyrosine in p40HERAK only after EGF pretreatment (Fig. 5b). Under the present experimental conditions, the tyrosine phosphorylation of p40HERAK resides at the limits of detection, which precludes a more quantitative analysis at this time. Nonetheless, the detection of phosphotyrosine on p40HERAK is remarkable, for this is a common regulatory feature ascribed to members of the mammalian MAPK/ERK family, as well as the yeast enzyme p40FUS3 (1). In addition to EGF-mediated tyrosine phosphorylation, the observed growth factor sensitivity, profile of immunoreactivity, and deduced substrate specificity (23) are all consistent with the assignment of p40HERAK as a novel member of the MAP kinase family.
In summary, we have identified a new member of the MAP kinase family of serindthreonine protein kinases that is physically associated with EGF-class receptors. This receptor-associated kinase phosphorylates the EGF receptor Thr-669 phosphorylation site motif with relatively high affinity. While the regulatory significance of this receptor phosphorylation has yet to be fully elucidated (20,35), this site-specific phosphorylation is predominant in vivo (19,201, and the corresponding site is also conserved in both HER2 (36) and HER3 (37). This particular domain within these growth factor receptors is in close proximity to the intrinsic tyrosine kinase ATP binding site, where covalent modification may well have subtle but important kinetic effects (35, 38). The finding that a similar and immunologically related protein kinase is associated with the EGF receptor and with HER2 is particularly interesting in that these receptors have been found to associate as heterodimers in cells which co-express both receptors (39). Alternatively, this novel EGF receptor-associated kinase may be representative of a new subfamily of receptor-associated M A P & that are specific for receptor subtype. Since both the EGF receptor and HER2 are characterized as proto-oncogenes, the abnormal expression and biological activity of which are closely correlated with a number of human neoplasms, determination of the functional role of the receptor-associated serindthreonine kinase in modulating receptor function and/or mitogenic signaling may provide new insights into the mechanisms of oncogenesis as well as a strategic locus for potential therapeutic intervention.