Regulation of human type II phosphatidylinositol kinase activity by ...

Vol. 269, No. 49, Issue of December 9, pp. 31243-31251, 1994 Printed in U.S.A.

T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Regulation of Human TypeI1 Phosphatidylinositol Kinase Activity by Epidermal Growth Factor-dependent Phosphorylation and Receptor Association* (Received for publication, April 7, 1994, and in revised form, October 5, 1994)

Andrea Kauffmann-ZehSO, Reinhard Klingefl, Gerda Endemannil,Michael D. Waterfield**, Reinhard WetzkerSSOO, and J. Justin HsuanSm From the protein Biochemistry Group, **Ludwig Institute for Cancer Research, UCL Medical School, 91 Riding House Street, London W l P 8Bl: United Kingdom, the $$Max-Planck Research Group “Growth Factor Signal Bansduction,” Drackendorfer Strape 1, IZnstitut fur Biochemie ZI, Friedrich-Schiller-UniversitatJena, Lobderstrasse 3, DO7740 Jena, Federal Republic of Germany, IlScios Nova Znc., Mountain View, California 94043,and the **Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6Bl: United Kingdom

Epidermal growth factor (EGF) stimulates phosphati- sponse to EGF, the receptor is able to phosphorylate itself on dylinositol (PtdIns) hydrolysis in many cell types by ef- multiple intracellular sites toward the carboxyl terminus, as fecting the specific interaction between the EGF recep- well as a range of heterologous substrates. The EGF receptor Cy. Several studies have belongs to the largefamily of receptor tyrosine kinases, all of tor and phospholipase suggested that PtdIns 4-kinase activity can also be regu- which appear to undergo tyrosinephosphorylation when actilated byEGF, but the mechanism of this stimulation was vated by their cognate ligand(s1. Specific subsets of molecules possessing SH2 (src-homology region2) domains, including unclear. We report here that EGF treatment of intact A431 cells increased the association of type I1 PtdIns phospholipaseC (PLC) y, PtdIns 3-kinase, Syp, GRB2, and kinase with the EGF receptor within 1 min at 37 “C. SHC, are able to bind certain phosphotyrosine residues on a Phosphorylation of immunoprecipitated EGF receptor range of receptor tyrosine kinases and thereby form signal also increased the association of PtdIns 4-kinase. Fur- transduction complexes on the intracellularface of the plasma thermore dephosphorylationof phosphoserine residues membrane (2,3).One such signalis a common response elicited on the stimulated receptor immune complex led to inac- by many different growth factors includingEGF and involves tivation of the bound PtdIns 4-kinase, while dephosphothestimulation of PtdIns hydrolysis (4, 5 ) . Earlystudies rylation of phosphotyrosine residues led to activation. showed that following treatment of A431 human epidermoid Unlike the stimulated activity measured in total cell and carcinoma cells with EGF, increased phospholipase C (PLC)y l plasma membranelysates, the changes in activity of the activity can be coprecipitated with the EGF receptor (6). The immunoprecipitateswereapparent at high substrate increased association is mediatedby binding of PLCyl tophosconcentration. Metabolic labeling was used to show that a 55-kDa phosphoserine and phosphotyrosine-contain- photyrosine residue 992 of the EGF receptor (7, 8), and both phosphorylation are required to stimuing protein comigrated with renatured PtdIns 4-kinase interaction and tyrosine late PLCyl activity(8).Similar mechanismsof association and activity on SDS-polyacrylamidegelelectrophoresis, while in vitro labeling revealed onlyserine phosphoryl- activation havebeen reported for the regulationof PtdIns 3-kiation. These data are discussed with reference to the nase by platelet-derived growth factor receptors (9). More redirect regulation of PtdIns 4-kinase by phosphorylation, cent results suggest that other autophosphorylation sites on association with PtdIns compartmentalization, and the formation of a the EGF receptor are also able to mediate the PLCy (10) and that theEGF receptor can also activate PLCy multienzyme signal transduction complex. independently of tyrosine phosphorylation (11). EGF has long been known to stimulate PtdInshydrolysis in The pleiotropic responses of cells to treatment with epider- epithelial andfibroblast cell types (4). Manystudies of changes mal growth factor (EGF)’ are induced by the activation of the in PtdIns turnover in responseto growth factors have focused EGF receptor, a 170-kDa transmembrane glycoprotein contain- on the stimulation of PLCyl activity as measured by the hy(1).In re- drolysis of PtdIns 4,5-bisphosphate. The increased hydrolysis ing an intracellular protein tyrosine kinase activity of PtdIns 4,5-bisphosphate following stimulation of PLCyl of inositol 1,4,5-trisphosphate * The costs of publication of this article were defrayed in part by the raises the cellular concentration be hereby marked payment of page charges. This article must therefore and diacylglycerol, which lead to the release of calcium ions “advertisement”in accordance with 18 U.S.C.Section 1734 solelyto from the endoplasmic reticulum and increased protein kinase C indicate this fact. activity, respectively. More recently EGF has been shown to 8 Fellow of CAPES-Brazilian Coordenadoria de Bolsas no Exterior. 4-phosphate 5-ki$8 Supported by grants from the DFG (We 1565/1-2and SFB197), the rapidly stimulate PtdIns 4-kinase and PtdIns British Council ARC Programme, and the Deutscher Akademischer nase activities inA431 cells (12-14) and transfectedB82 mouse Austauschdienst (DAAD). fibroblasts (15). These two kinases sequentially phosphorylate lill Supported by the British Council ARC Programme and the DAAD. the D-4 and D-5 positions of the inositol residue of PtdIns, To whom all correspondence should be addressed: Head of Protein Biochemistry, LudwigInstitute for Cancer Research, UCL Medical School, thereby generating PtdIns 4,5-bisphosphate, the main substrate for PLC enzymes. Increased PtdIns4-kinase activity has 91 Riding House St., London, WClE 6BT. ‘The abbreviations used are: EGF, epidermal growth factor; PLC, also beenreported following stimulation of the c-erbB2 receptor phosphatidylinositol-specific lipase C ; PtdIns, phosphatidylinositol; tyrosine kinase in overexpressing breast cancer cells (16). HowgPtdIns, deacylated PtdIns; PITP, PtdIns and phosphatidylcholine-speever the mechanism of this activation remained unclear and in cific transfer protein; HPLC, high performance liquid chromatography; following the DMEM, Dulbecco’s modified Eagle medium; PVDF, polyvinylidene di- particularwhether or not productdepletion fluoride; PAGE, polyacrylamide gel electrophoresis. stimulation of PLC activity could alone account for the previ-

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PtdIns 4-Kinase Regulation by EGF

ously reported observations. In this regard the cytosolic PtdIns leupeptin, 1mM phenylmethylsulfonyl fluoride, and 10 pg/ml benzamitransfer protein (PITP) was recently shown to be a necessary dine (lysis buffer)and allowed to stand for 10 minon a rocking platform component of the GTP and ATP-dependent stimulation of PLCp at 4 "C. Lysates wererecovered with a rubber policeman and cleared by activity in permeabilized HL60 cells. The requirement for Pt- centrifuged for 10 min at 15,000 x g at 4 "C and by incubation with protein A-Sepharose CL4B for 30 min at 4 "C. Cell lysates were incudIns transfer and ATP suggests that at least in this situation bated with 4C5G (30), PY20, 1G2, or R1 (31)monoclonal antibodies at the increased generation of second messengers requires both 4 "C for 2 h on a rotary wheel. Immunocomplexes were formed with stimulated PtdIns phosphorylation and stimulated phosphoi- protein A-Sepharose CL4B for 1 h. Precipitates were washed extensively with lysis buffer and once with PtdIns4-kinase assay buffer (see nositidase activities (17). Mammalian PtdIns kinases have previously been divided below) when PtdIns 4-kinase assays were performed or with protein into threeclasses (18, 19). Type I enzymes possess PtdIns 3-ki- kinase assay buffer(see below) for in vitro protein kinase assays. Phenylphosphate elutions were carried out on 1G2 immunoprecipitates by nase activity and are heterodimers of 85- and 110-kDa sub- incubating thepellets with 15 mM phenylphosphate for 30 min a t 4 "C units, both of which have been purified and cloned (20-22). in lysis buffer and recovering the eluates aftercentrifugation. While this enzyme activity clearly associates with many actiAssay of PtdIns 4-Kinase Activity in Immunoprecipitates-PtdIns vated growth factor receptors, the role of 3-phosphorylated Pt- 4-kinaseactivitywas measured as described previously with minor dIns and PtdIns phosphates remains be to shown. However, the modifications (32). Briefly, samples were incubated with 25 mM Tris/ identification of the PtdIns 3-kinase activity of the Saccharo- HCl, pH 7.5, 10 mM MgCl,, 70 PM ATP, 1.5% Triton X-100 (PtdIns 4-kinase assay buffer) containing200 p~ PtdIns (approximately 0.8 mol myces cerevisiae ups34 protein (23) suggests that this enzy- %) and 5 pCi of [Y-~~PIATP for 20 min at 37 "C. Reactions were stopped matic activity may be involved in the formation and targeting by the addition of 1ml of hexane/isopropanol (13:7, v:v) and 0.2 ml of 2 of intracellular vesicles (24). The type I1 and I11 enzymes both M KCl, concentrated HCl (8:0.25, v:v). Tubes were vortexed, and 0.5 ml containing 0.5 ml of possess PtdIns 4-kinase activity, but differ in their apparent of the organic phase was transferred to fresh tubes size and enzymatic properties, including their K,,, for ATP and 0.1 M HCl. After vortexing, the radioactive components of the final organic phase were identified by TLC and quantitated by liquid scinPtdIns, and their sensitivityinhibition to by adenosine (19,25). tillation counting (32). PtdIns 4-kinase activity was assayed following It is the widely expressed type I1 activity that appears to be SDS-PAGE and renaturation as previously described (32). sensitive to growth factors (25). While a S. cereuisiae PtdIns Phosphorylation of Proteins in Vitro-Reactions were carried out in 4-kinase with intermediate properties has recently beencloned protein kinase assay buffer (25 mM Tris/HCl, pH 7.5, 10 m MgCI, and (261, neither type of mammalian enzyme has yet been cloned, 0.2 mM EGTA) in thepresence of 50-250 PM final ATP concentration. For although many purifications of the type I1 enzyme have been radioactive labeling, 1-2 pCi of [7i('P]ATP was included. Phosphorylation was carried out a t 30 "C for 5-30 min. Reactions were stopped by reported (25). the addition of 2 x sample buffer and boiled for 5 min. Samples were In addition to the plasma membrane activity, PtdIns 4 4 - separated by SDS-PAGE, and gels were either stainedor electroblotted nase activities have been found in many subcellular organelle onto PVDF filters. Samples were exposed to Amersham MP film or membranes (251, but little is known about the function of the Kodak XAR film at -70 "C. Phosphorylations were analyzedby excising enzyme in these compartments.Nevertheless, despite numer- bands of interest as described below. Dephosphorylation Reactions-Bacterial alkaline phosphatase was ous different studies, the functional compartmentalization of used for nonspecific dephosphorylations. These reactions were carried PtdIns metabolism remains poorly understood (27-29). out by incubation with approximately 5 units of phosphatase in 50 m~ We report here our studies on the mechanism of type I1 TrisiHC1, pH 8.8, 50 mM NaC1, 1mM dithiothreitol, 1pdml aprotinin, 1 RdIns kinase activation following the treatment of A431 cells pdml leupeptin, 1 pg/ml pepstatin, and 1 unit'ml a-macroglobulin for with EGF. Evidence is presented t o show that this PtdIns ki- 30 min at 20 "C. Protein phosphotyrosine phosphatase 67E was usedfor the specific dephosphorylation of phosphotyrosine residues. Approxinase is phosphorylated in immunoprecipitates and in intact cells on serine and tyrosine residues concomitant with stimu- mately 10 units were used for each dephosphorylation reaction, performed as described elsewhere (33). Dephosphorylation of phospholation of the enzyme activity. Furthermore phosphatase treat- serine and phosphothreonine residues was performed using PP2A a t ment canabolish this activation, and activity can be restored by 30 "C for 30 min in 20 mM Hepes, pH 7 . 5 , l mM MnCl,, 10 mM MgCl,, 1 rephosphorylation. These results clearly suggest a direct mech- mM dithiothreitol. Metabolic Radiolabeling"A431 cells grown as above were transanism and regulatory importancefor the previously observed ferred to phosphate-free medium containing 0.5% dialyzed fetal calf activation of the human PtdIns4-kinase. EXPERIMENTALPROCEDURES Material~-[y-~~P]ATP, [32P]phosphate,1251-proteinA, and Hyperfdm were from Amersham International (Bucks, UK). Phosphatidylinositol, Triton X-100, and protease inhibitors were from Sigma (Dorset, UK). TLC plates (Silica gel 60 without indicator) were obtained from Merck (Leics, UK). Protein A-Sepharose CL4B and bacterial alkaline phosphatase were from Pharmacia Biotech Inc. (Herts, UK). Antiphosphotyrosine antibody PY20 was purchased from ICN (Bucks, UK) and 1G2agarose was from Oncogene Science, Cambridge Bioscience (Cambridge, UK). All monoclonal antibodies were purified using protein Aaffinitychromatography. Immobilon-P (polyvinylidene difluoride) membrane was from Millipore (Herts, UK). DMEM, fetal calf serum, penicillin, and streptomycin were from Life Technologies, Inc. (Scotland, UK). Protein tyrosine phosphatase 67E,proteinserine/ threonine phosphatase 2A (PPBA), and anti-SHCantibody were kindly provided by Dr.P. Vicendo (Universitb Paul Sabatier,Toulouse, France), Prof. P. Cohen (University of Dundee, UK), and Dr. T. Pawson (Mount Sinai Hospital, Toronto, Canada). Immunoprecipitations and Phenylphosphate Elution-A431 cells were grown in DMEM supplemented with 10%fetal calf serum. When cells reached 50% confluence, they were starved for 24 h in DMEM containing 0.5% fetal calf serum before EGF stimulation for 2 min at 37 "C. Cells were washed three times withice-cold phosphate-buffered saline and lysed with ice-cold 25 mM TrisiHCl, pH 7.4, 150 mM NaCl, 2 mM EGTA, 1%Triton X-100, 0.25 mM sodium orthovanadate, 10 pg/ml

serum and labeled with 32P-labeledinorganic phosphate at 1 mCi/ml. Approximately 16 h later, cells were treated with or without EGF and immunoprecipitated with 1G2, R1, or 4C5G antibodies as described above. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and detected by autoradiography. Phosphoamino Acid Analysis-32P-Phosphorylated proteins were eluted from the excised gel bands and subjected to partial acid hydrolysis and two-dimensional electrophoretic separation of 32P-phosphorylated amino acids at pH 1.9 and3.5 (34). Immunoblotting-Immunoprecipitates were produced as described above, analyzed by SDS-PAGE and electrophoretically transferred to PVDF membranes. Immunoblotting was performed with the anti-SHC antibody (35), antiphosphotyrosine antibody PY20 or with the anti-EGF receptor antibody 9A (36). Bound antibodies were detected using ''Ilabeled protein A. Lipid Analysis-Identification of phosphoinositides was performed using the HPLC technique described by Auger et al. (37). In summary, lipids were recovered from TLC plates, deacylated, and injected in a Partisphere SAX column (Whatman Int.,Kent, UK). A [3HlPtdIns 4-phosphate standard was used to confirm the identity of the enzyme product. RESULTS

Association of PtdZns 4-Kinase withEGF Receptor-A previous report hassuggested that type I1 PtdIns kinase (hereafter otherwise) can referred to as PtdIns 4-kinase unless stated

by EGF

Regulation 4-Kinase PtdIns bind to the juxtamembraneregion of the activated EGFreceptor (15). While this phenomenon was EGF dependent and was not observed with a carboxyl-terminally deleted receptor, the synthetic peptidesused intheseexperiments (YLRRRHIVRKRTLRRLLQERELVE and VRKRTLRRL, human EGF receptor residues 643-666 and 650-658, respectively) are extremely basic and this association could simply be the result of nonspecific, ionic interactions rather than specific binding to this particular amino acid sequence, particularly as PtdIns 4-kinase activity binds efficiently to a MonoQ anion exchange column a t pH 8.0 (32), isknown t o be activated by numerous polycations a t pH 7.4 (38), and may be phosphorylated in response to EGF (seebelow). An outstanding question is whether PtdIns 4-kinase is recruited to the receptor or whether already bound PtdIns 4-kinase is stimulated by receptor activation. To help clarify this point we took advantage of the monoclonal antibody 4C5G which specifically recognizes and inhibitstype I1 but not type I or I11 PtdIns kinase activities (30). A maximum of approximately 60-70% of the total PtdIns 4-kinase activity of total A431 cell lysates can be inhibited by titration of 4C5G (data not shown), which issimilartoearlierresults (30). Thisantibody does not recognize the denatured enzyme using immunoblotting. We first tested whether this antibody could immunoprecipitate PtdIns 4-kinase from A431 human epidermoid carcinoma cells by exploiting the ability of PtdIns 4-kinase to renature following SDS-PAGE. A431 cell lysates were immunoprecipitated with 4C5G antibody and washed extensively. The immunoprecipitates were subjected to SDS-PAGE a t 4 "C and the lanes containing the samples cut into approximately 1-mm gel slices, macerated and subjected to PtdIns 4-kinase assays at 20 "C. As illustrated in Fig. lA the renatured activity was indeed recovered and migratedwith the expected molecular mass of 55 kDa (39).Parallel immunoprecipitations performed using EGF-R1, which specifically recognizes the native human EGF receptor (31),and antiphosphotyrosine antibodies showed that stimulation with EGF increasedthe amountof PtdIns 4-kinase activity measured in the immune complex by approximately 3-5-fold (Fig. 1 B ) . Only a small fraction (