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• W2018575189 abstract "FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC λ when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC ζ, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC λ is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300–508) and SNT2 (amino acids 281–492), an isoform bearing 50% identity to FRS2, interacted with PKC λ at a region (amino acids 240–562) that encompasses the catalytic domain.In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC λ or ζ. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC λ results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its “closed” wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC λ mutant, suggesting that the activation of PKC λ is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways. FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC λ when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC ζ, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC λ is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300–508) and SNT2 (amino acids 281–492), an isoform bearing 50% identity to FRS2, interacted with PKC λ at a region (amino acids 240–562) that encompasses the catalytic domain.In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC λ or ζ. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC λ results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its “closed” wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC λ mutant, suggesting that the activation of PKC λ is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways. epidermal growth factor FGF receptor substrate-2 growth factor receptor-binding protein-2 Src homology domain-2 phosphotyrosine binding domain fibroblast growth factor basic fibroblast growth factor FGF receptor-1 platelet-derived growth factor glutathioneS-transferase polyacrylamide gel electrophoresis myelin basic protein protein kinase C protein kinase A Suc1-associated neurotrophic factor-induced tyrosine phosphorylation target insulin receptor substrate Src homology containing tyrosine phosphatase-2 heterogenous ribonucleoprotein A1 atypical PKC conventional PKCs novel PKCs hemagglutinin mitogen-activated protein polymerase chain reaction phosphoinositide-dependent protein kinase 1 phosphatidylinositol 3-kinase protein-tyrosine phosphatase Fibroblast growth factor receptors are members of the receptor-tyrosine kinase family (1Jaye M. Schlessinger J. Dione C.A. Biochim. Biophys. Acta. 1992; 1135: 185-199Crossref PubMed Scopus (594) Google Scholar). In contrast to other growth factor receptors such as those for EGF1 and PDGF, FGF receptors are poorly auto-phosphorylated upon ligand binding. Instead, a 90-kDa protein called SNT1 or FGF receptor substrate-2 (FRS2) (2Kouhara H. Hadari Y.R. Spivak T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 3Rabin S.J. Clegon V. Kaplan D.R. Mol. Cell. Biol. 1993; 13: 2203-2213Crossref PubMed Scopus (173) Google Scholar) is phosphorylated at multiple tyrosine sites. FRS2 has also been reported to be serine/threonine-phosphorylated in FGF-treated cell lysates (4Wang J.K. Xu H. Li H.C. Goldfarb M. Oncogene. 1996; 13: 721-729PubMed Google Scholar). SNT2, a recently identified isoform of FRS2, has about 50% identity to FRS2 (5Xu H. Lee K.W. Goldfarb M. J. Biol. Chem. 1998; 273: 17987-17990Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) mainly at the N- and C-terminal ends. FRS2 and SNT2 possess a myristoylation site and a PTB domain at the N terminus. The PTB domain is responsible for the direct interaction of FRS2 and SNT1 with the juxtamembrane region of the FGFR in a phosphotyrosine-independent manner. Deletion of the PTB domain of both proteins abrogates the association and tyrosine phosphorylation of FRS2 and SNT2 by FGF receptors (5Xu H. Lee K.W. Goldfarb M. J. Biol. Chem. 1998; 273: 17987-17990Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6Lin H.Y. Xu J.S. Ischenko I. Ornitz D.M. Halegoua S. Hayman M. Mol. Cell. Biol. 1998; 18: 3762-3770Crossref PubMed Scopus (60) Google Scholar). FRS2 and SNT2 substitute for their receptors as docking proteins, a role similar to that of insulin receptor substrate (IRS) in insulin signaling (7Yenush L. White M.F. BioEssays. 1997; 19: 491-500Crossref PubMed Scopus (251) Google Scholar). To date, two important signaling proteins, Grb2 and SHP-2, have been reported to bind directly to tyrosine-phosphorylated FRS2 (2Kouhara H. Hadari Y.R. Spivak T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 8Hadari Y.R. Kouhara H. Lax I. Schlessinger J. Mol. Cell. Biol. 1998; 18: 3966-3973Crossref PubMed Scopus (268) Google Scholar). Grb2 is an adapter protein best known for its role in linking receptor tyrosine kinases to the Ras pathway via the guanine nucleotide-releasing factor Sos (9Schlessinger J. Curr. Opin. Genet. & Dev. 1994; 4: 25-30Crossref PubMed Scopus (400) Google Scholar). The binding of Grb2 to FRS2 occurs via the interaction of the SH2 domain of Grb2 with some or all of the potentially phosphorylated tyrosine residues at Tyr-196, Tyr-306, Tyr-349, and Tyr-392 on FRS2. Mutational studies showed that when the tyrosine residues at all 4 sites were changed to phenylalanine, the downstream MAP kinase activation was significantly reduced (2Kouhara H. Hadari Y.R. Spivak T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). SHP-2 is a tyrosine phosphatase whose activity has been proposed to be necessary for cell growth and proliferation (10Bennett A.M. Hausdorff S.F. O'Reilly A.M. Freeman R.M. Neel B.G. Mol. Cell. Biol. 1996; 16: 1189-1202Crossref PubMed Scopus (226) Google Scholar, 11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). When cells are stimulated with growth factors such as PDGF, SHP-2 is tyrosine-phosphorylated and binds to the SH2 domain of Grb2 (12Li W. Nishimura R. Kashisian A. Batzer A.G. Kim W.J.H. Cooper J.A. Schlessinger J. Mol. Cell. Biol. 1994; 14: 509-517Crossref PubMed Google Scholar). SHP-2 also binds to the activated receptors via its own SH2 domain (12Li W. Nishimura R. Kashisian A. Batzer A.G. Kim W.J.H. Cooper J.A. Schlessinger J. Mol. Cell. Biol. 1994; 14: 509-517Crossref PubMed Google Scholar). As a result, SHP-2 functions not only as a phosphatase but also serves as an adapter protein recruiting Grb2 to the receptors. Recently, SHP-2 has been reported to bind directly to tyrosine-phosphorylated FRS2 through its N-terminal SH2 domain (8Hadari Y.R. Kouhara H. Lax I. Schlessinger J. Mol. Cell. Biol. 1998; 18: 3966-3973Crossref PubMed Scopus (268) Google Scholar). The association of SHP-2 with FRS2 and the activation of SHP-2 are essential for a sustained MAP kinase response as well as for the potentiation of FGF-induced neurite outgrowth in PC12 cells (8Hadari Y.R. Kouhara H. Lax I. Schlessinger J. Mol. Cell. Biol. 1998; 18: 3966-3973Crossref PubMed Scopus (268) Google Scholar). Hence, by recruiting Grb2 and SHP-2, FRS2 plays a crucial role in linking the FGF receptors to the Ras/MAP kinase pathway. Apart from the Grb2 and SHP-2 proteins, the activity of the atypical PKCs (aPKCs) is necessary for mitogenic signaling via the MAP kinase cascade (13Edurne B. Diaz-Meco M.T. Dominguez I. Municio M.M. Sanz L. Chapkin S. Moscat J. Cell. 1993; 74: 555-563Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 14Edurne B. Diaz-Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (251) Google Scholar). There are two members in the aPKC subfamily, PKC λ and PKC ζ, and they share more than 75% identity. PKCs have been subdivided into 3 subfamilies, and they are distinguished by their lipid activation profiles. Conventional PKCs (cPKCs e.g.α, β, and γ) are activated by diacylglycerol and calcium; novel PKCs (nPKCs e.g. δ, ε, η, and θ) do not respond to calcium but require diacylglycerol for their activation; and aPKCs are not activated by either diacylglycerol or calcium. It has been shown that MAP kinase and MEK are activated in vivo by an active mutant of PKC ζ, and a kinase-defective dominant negative mutant of PKC ζ impairs the activation of both MEK and MAP kinase by serum and tumor necrosis factor (14Edurne B. Diaz-Meco M.T. Lozano J. Frutos S. Municio M.M. Sanchez P. Sanz L. Moscat J. EMBO J. 1995; 14: 6157-6163Crossref PubMed Scopus (251) Google Scholar). However, whereas Grb2 and SHP-2 lie upstream of Ras, PKC ζ can bind to and act as a direct effector of Ras (15Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar). This is consistent with the observation that expression of a dominant negative mutant of Ras (Asn-17) severely impairs the activation of PKC ζ by mitogens such as PDGF in mouse fibroblasts (15Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar). A few groups of proteins that are either regulators or substrates of aPKCs bind to the members in this subfamily. In the first group, a protein called Zeta-InteractingProtein (ZIP) binds specifically to the regulatory domain of PKC ζ (16Plus A. Schimdt S. Grawe F. Stabel S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6191-6196Crossref PubMed Scopus (193) Google Scholar), whereas Lambda-InteractingProtein (LIP) binds specifically to the regulatory domain of PKC λ resulting in an activation of the kinase (17Diaz-Meco M.T. Munico M.M. Sanchez P. Lozano J. Moscat J. Mol. Cell. Biol. 1996; 16: 105-114Crossref PubMed Google Scholar). The Par-4 protein also binds to the regulatory domain of PKC λ and PKC ζ but inhibits their activity (18Diaz-Meco M.T. Municio M.M. Frutos S. Sanchez P. Lozano J. Sanz L. Moscat J. Cell. 1996; 86: 777-786Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The second group comprises proteins like heterogeneous ribonucleoprotein A1 protein that has been found to bind to the kinase domain of PKC ζ in yeast two-hybrid screening and is a specific substrate of the aPKCs (19Municio M.M. Lozano J. Sanchez P. Moscat J. Diaz-Meco M.T. J. Biol. Chem. 1995; 270: 15884-15891Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). We have been studying p75, a phosphotyrosine protein that is dephosphorylated and dissociates from Grb2 upon growth factor stimulation (27Lim Y.P. Low B.C. Ong S.H. Guy G.R. J. Biol. Chem. 1997; 272: 29892-29898Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). In our attempt to identify p75 by immunoprecipitating phosphotyrosyl proteins that are about 75-kDa in molecular mass, we observed that a 90-kDa tyrosine-phosphorylated protein, p90, associates specifically with members of the aPKC subfamily but not with other PKC family members. In this report, we identified the p90 protein as FRS2. We have also characterized the factors that regulate its association with the aPKCs. We propose that FRS2 plays an important role in the targeting of activated PKC λ or ζ to the plasma membrane. Thus FRS2 may constitute a third group of proteins that bind to the aPKCs and localize them in specific subcellular compartments. The recruitment of aPKCs by FRS2 to the cell-surface membrane may be an important event contributing to the regulation of the aPKC activity. Monoclonal antibodies against phosphotyrosine (PY20), Grb2, SHP-2, and PKC λ were purchased from Transduction Laboratories (Lexington, KY). Polyclonal antibodies against PKC α, δ, and PKC λ/ζ were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against FRS2 (A872) were raised against amino acids (residues 491–506) and produced by Neosystem Laboratoire (Strasbourg, France). Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were from Sigma, and protein A/G plus agarose was from Santa Cruz Biotechnology. Anti-activation domain and anti-binding domain antibodies are fromCLONTECH (Palo Alto, CA). Recombinant human EGF and PDGF were from Sigma, and basic FGF (bFGF) was from Roche Molecular Biochemicals (Mannheim, FRG). PKC ζ, PKC α, PKC δ, and PKA purified enzymes were from Life Technologies, Inc. Swiss 3T3 fibroblasts (ATCC CCL92, Rockville, MD) were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 2 mmglutamine, 10 mm HEPES, pH 7.4, and 100 units/ml penicillin and streptomycin. Human 293T kidney epithelial cells were grown in 150-mm culture dishes with RPMI medium supplemented with 10% fetal bovine serum (HyClone Laboratories), 2 mm glutamine, 10 mm HEPES, pH 7.4, and 100 units/ml penicillin and streptomycin. When the cells were about 80–90% confluent, the medium was aspirated, and the cells were washed and maintained for another 18–24 h in serum-free medium. Various growth factors were added to the quiescent cells prior to aspiration of the medium. The cells were then washed rapidly in cold phosphate-buffered saline and lysed in 500 μl of lysis buffer containing 0.5% Nonidet P-40, 20 mmTris-HCl, pH 7.3, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1 mm sodium orthovanadate, and a mixture of protease inhibitors (Roche Molecular Biochemicals) added according to the manufacturer's instructions. The cell lysates were spun at 11,000 × g for 5 min at 4 °C, and the supernatants were used for subsequent analyses. The protein concentrations of all cell lysates were normalized after estimation of their protein content using a BCA protein assay kit from Pierce. PKC λ/ι cDNA and HA-tagged PKC ζ in pCDNA3 were kind gifts from Dr. Jorge Moscat (Universidad Autonoma de Madrid, Spain). PKC βII cDNA and PKC δ cDNAs were from Dr. Alexandra Newton (University of California, San Diego) and Dr. Li Weiqun (National Cancer Institute, Bethesda), respectively. cDNAs encoding the full-length PKC λ, PKC λ fragment A (amino acids 1–239), PKC λ fragment B (amino acids 240–586), PKC ζ fragment B (amino acids 240–592), PKC δ fragment B (amino acids 354–701), and PKC βII fragment B (amino acids 345–673) were obtained by PCR. These inserts were introduced into pGEX4T1 vector for the expression of GST fusion proteins in bacterial cells. FRS2 cDNA was obtained first by reverse transcription from mRNA extracted from Swiss 3T3 cells. PCR was then carried out with the following primers, which were designed based on the published sequence of FRS2 (2Kouhara H. Hadari Y.R. Spivak T. Schilling J. Bar-Sagi D. Lax I. Schlessinger J. Cell. 1997; 89: 693-702Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar), to obtain the full-length cDNA as follows: (forward) 5′ CGC GGA TCC GCG ATG GGT AGC TGT TGT AGC TGT CC 3′ and (reverse) 5′ CG GCGG CCGC TCA CAT GGG CAG GTC AGT ACT ATT G 3′. TheBamHI/NotI insert was introduced into pGEX4T1 and pXJ40HA for the expression of GST fusion protein in bacteria cells and HA-tagged proteins in mammalian cells, respectively. The expressed proteins were partially microsequenced and shown to be authentic. The FRS2 fragments X (amino acids 1–152), Y (amino acids 153–300), Z (amino acids 301–508), XY (amino acids 1–300), and YZ (amino acids 153–508) were obtained by PCR using the full-length FRS2 cDNA as template. All the inserts were cloned into pGEX4T1 for the expression of GST fusion proteins. Human SNT2 cDNA was a kind gift from Dr. Mitchell Goldfarb (Mount Sinai School of Medicine, New York). cDNA encoding the fragment Z (amino acids 281–492) of SNT2 was obtained by PCR and cloned into pGEX4T1 for the production of GST fusion proteins. The cDNA for human FGF receptor 1 (Flg) was a kind gift from Dr. Lena Claesson-Welsh (Ludwig Institute for Cancer Research, Uppsala, Sweden). cDNA encoding the cytoplasmic domain (amino acids 398–822) of Flg was obtained by PCR and cloned into pXJ40Flag for the expression of Flag-tagged cytosolic Flg in mammalian cells. For yeast two-hybrid screening, cDNAs encoding the full-length fragment A (amino acids 1–239) and fragment B (amino acids 240–586) of PKC λ were obtained by PCR as described above and introduced into pAS vector suitable for yeast transformation and expression of Gal4-binding domain fusion protein. SHP-2 cDNA was a kind gift from Dr. Tony Pawson (Mount Sinai Hospital, Ontario, Canada). Full-length FRS2 and SHP-2 were subcloned into pACT vector for yeast expression of Gal4 activation domain fusion proteins. cDNAs for the tandem SH2 domains (amino acids 1–213) and PTP catalytic domain (amino acids 214–603) of SHP-2 were obtained via PCR, and the inserts were also cloned into the pACT vector. Mutation of alanine to glutamate A120E in the pseudo-substrate site of PKC λ was carried out using the QuickChange mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's instruction. The template used was wild-type full-length PKC λ in pGEX4T1 and pXJ40HA. The primers used were as follows 5′ CCG GAG AGG GGA ACG CCG TGG GAG 3′ and 5′ CTC CAC CGG CGT TCC CCT CTC CGG 3′. The products were sequenced and verified to be correct. Human 293T kidney epithelial cells were grown in 100-mm culture dishes as described above. Cells that were about 90% confluent were used for transfection. For single or co-transfections, 15 μg of each DNA followed by 4.5 μl/μg DNA of TfX 50 from Promega (Madison, WI) were added to 6 ml of serum-free RPMI and incubated at room temperature for 15 min. The transfection mix was then added to cells prewashed with serum-free medium and left at 37 °C for 1 h. After this, 12 ml of RPMI supplemented with 10% fetal bovine serum was added, and the cells were left to recover for 48 h. The cells were lysed with RIPA buffer (50 mm Tris-HCl, pH 7.3, 150 mm NaCl, 0.25 mm EDTA, 1% sodium deoxycholate, 1% Triton X-100, 1 mm sodium orthovanadate, and a mixture of protease inhibitors from Roche Molecular Biochemicals) and processed as described under “Cell Lines, Cell Stimulation, and Cell Lysis.” All the constructs for the production of GST fusion proteins were transformed into DH5α cells. The transformed cells were grown in 1 liter of LB + ampicillin (50 μg/ml) medium and incubated at 37 °C with shaking (220 rpm) to anA 600 of about 0.3. These cells were then induced with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside at room temperature overnight. The cells were spun down and frozen at −80 °C. The cell pellet was then left to thaw on ice, and 10 ml of lysis buffer (phosphate-buffered saline, 1% Triton X-100, 1 mm dithiothreitol, and a mixture of protease inhibitors from Roche Molecular Biochemicals) was added to the cell. The cell suspension was subsequently sonicated for a total of 12 pulses of 15 s with a 30-s pause between each pulse. The lysates were centrifuged and supernatants were incubated with glutathione beads overnight at 4 °C to purify the GST fusion proteins. Quiescent or activated cells were lysed as described above, and an equal volume of 2× precipitation buffer (20 mm Tris-HCl, pH 7.4, 300 mm NaCl, 2% Triton X-100, 2 mm EDTA, 2 mm EGTA, and 1% Nonidet P-40) was added to the cell lysate. For immunoprecipitation, 2.5 μg of the appropriate antibodies were added to the diluted cell lysate and incubated for 1 h or overnight at 4 °C. 2.5 μg of secondary antibodies conjugated to agarose was added to capture the immunocomplex for 1 h or overnight at 4 °C. In the depletion studies, the immunoprecipitation described above was repeated 5 times, each for 1.5 h. After washing, the immunoprecipitates were pooled together and resolved by SDS-PAGE. For in vitro binding assays with GST fusion proteins, 10 μg of the GST fusion proteins were incubated with the lysates for 1 h or overnight at 4 °C. All the beads were washed 3 times with 1× precipitation buffer, and the bound proteins were eluted with 2× Laemmli buffer before separation by SDS-PAGE. The various constructs of PKC λ (full length, fragment A, and fragment B) in pAS were sequenced and verified to be correct before they were introduced into yeast strain 190 using the yeast transformation kit fromCLONTECH (Palo Alto, CA). The transformed yeast were grown in selective media SD-Trp at 30 °C until colonies appeared. Single transformants then underwent a second round of transformation with the pACT vectors containing full-length FRS2, full-length SHP-2, SH2 domains, or PTP catalytic domain of SHP-2. Successful dual transformants were selected on SD-Trp/Leu, and LacZ blue assays were carried out, according to the manufacturer's instructions, to detect for protein interactions in the yeast. Yeast transformed with pCL1 expressing functional Gal4 protein turned blue between 0.5 and 1 h. This serves as a positive control for the LacZ assay. Colonies turning blue after 8 h were considered negatives according to the manufacturer's instruction. The dual transformants were also analyzed for the expression of the various proteins by first growing them in SD-Trp/Leu liquid medium. The yeast was lysed according to the transformation kit manufacturer's instructions, and the lysates were separated on SDS-PAGE. Following Western blotting, the various proteins were detected by probing with the appropriate antibodies. For activation studies of aPKCs by growth factors, Swiss 3T3 cells were either untreated or stimulated with bFGF at 20 ng/ml for 10 min. After the cells were lysed, immunoprecipitations of aPKC and subsequent in vitro kinase assays were carried out as described elsewhere (4Wang J.K. Xu H. Li H.C. Goldfarb M. Oncogene. 1996; 13: 721-729PubMed Google Scholar). In vitrokinase assays were also carried out either with purified PKA, PKC ζ, PKC α, or PKC δ enzyme purchased from Life Technologies, Inc. HA-tagged A120E PKC λ mutant and HA-tagged PKC λ fragment B (containing the kinase domain) were also used as a source of kinase activities. In cases where the HA-tagged PKC λ proteins were used, immunoprecipitations using HA antibodies were carried out before the kinase assays were performed. GST full-length FRS2 or GST-FRS2 fragment Z was tested as substrate for aPKCs, and hnRNPA1, a gift from Dr. Jorge Moscat (Universidad Autonoma de Madrid, Spain), and MBP were used as positive controls. All aPKCs reactions were carried out at 30 °C for 30 min in 20 μl of buffer (35 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 0.5 mm EGTA, 0.1 mm CaCl2, and 1 mm phenylphosphate) containing 50 ng of enzymes, 2 μg of substrate, 5 μCi of [γ-32P]ATP (Amersham Pharmacia Biotech), and 50 μm of ATP. The reactions for the PKC α and PKC δ were carried out in 20 μl of buffer (20 mm HEPES, pH 7.4, 1.5 mm CaCl2, 1 mm dithiothreitol, and 10 mm MgCl2) containing 50 ng of enzymes, 50 μg/ml sonicated phosphatidylserine, 2 μg of substrates, 5 μCi of [γ-32P]ATP (Amersham Pharmacia Biotech), and 50 μm of ATP. The reaction was stopped by boiling with an equal volume of 2× Laemmli buffer, and the proteins were separated on SDS-PAGE. The gel was dried and subjected to autoradiography. Our laboratory has been characterizing p75 and its association with Grb2. In quiescent cells, p75 is tyrosine-phosphorylated and binds to the SH2 domain of Grb2. Upon stimulation with growth factors including FGF, p75 is dephosphorylated and dissociates from Grb2 (27Lim Y.P. Low B.C. Ong S.H. Guy G.R. J. Biol. Chem. 1997; 272: 29892-29898Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). We were keen to identify p75 and decided to test existing phosphotyrosyl proteins that are about 75 kDa for dephosphorylation upon FGF treatment. One of the candidates that we selected was PKC δ, a member of the nPKC subfamily which is about 78 kDa and is the only PKC member currently known to be tyrosine-phosphorylated (33Li W. Mischak H. Yu J.C. Wang L.M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar). We therefore set out to investigate whether PKC δ is tyrosine-phosphorylated in quiescent cells. Representative members, namely PKC α and PKC λ, from the cPKC and aPKC subfamilies, respectively, were also included for comparison. Preliminary studies in our laboratory have shown that two FGF-responsive cell lines, Swiss 3T3 and PC12 cells, expressed all the three PKCs of interest. Swiss 3T3 cells were chosen for further experiments because they respond better to bFGF than PC12 cells. To examine whether PKC δ, PKC α, or PKC λ could be the p75 that undergoes dephosphorylation upon growth factor stimulation, immunoprecipitation of the various PKCs was carried out on lysates on Swiss 3T3 cells that were either untreated or stimulated with bFGF. The immunoprecipitates were resolved by SDS-PAGE and Western blotted. The membrane was probed with phosphotyrosine antibodies to detect for the presence of tyrosine-phosphorylated PKCs. None of the PKCs was tyrosine-phosphorylated in the lysates of quiescent cells (Fig.1 A, upper panel). The blot was stripped and re-probed either with PKC α, PKC δ, or PKC λ antibodies to show that the individual PKCs had been immunoprecipitated (Fig. 1 A, lower panel). We conclude that none of the PKCs tested are likely to be p75. In these experiments, we noted that a 90-kDa tyrosine-phosphorylated protein, similar to an FGF-specific p90 protein that has been reported to bind Grb2 (34Klint P. Kanda S. Claesson-Welsh L. J. Biol. Chem. 1995; 270: 23337-23344Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), was co-immunoprecipitated with PKC λ (Fig.1 A, upper panel). Neither PKC δ nor PKC α co-precipitated this tyrosine-phosphorylated protein significantly when compared with PKC λ in the lysates from bFGF-stimulated cells. We therefore decided to investigate this apparently specific association. It is possible that in the experiments carried out above, differential amounts of the various PKCs were immunoprecipitated by the antibodies due to the different affinities of the individual antibodies for their respective PKCs. The apparently larger amount of p90 co-immunoprecipitated with PKC λ may due to higher amounts of PKC λ immunoprecipitated compared with the other PKCs. Hence, it was necessary to ensure that the majority (>80%) of each PKC was immunoprecipitated. Preliminary optimization showed that five successive rounds of immunoprecipitation were enough to deplete 80% or more of the various PKCs (data not shown). Therefore, five rounds of immunoprecipitation of PKC λ, PKC α, and PKC δ (as described under “Experimental Procedures”) were carried out on lysates from Swiss 3T3 cells that have been stimulated with bFGF. The immunoprecipitates were pooled, resolved by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The membrane was then probed with phosphotyrosine antibodies to detect p90. Fig. 1 B, top panel, shows that p90 co-immunoprecipitated only with PKC λ. The blot was stripped and cut between lanes and probed for the various PKCs immunoprecipitated (Fig. 1 B, middle panel). The amounts of the various PKCs present in the lysates before and after multiple immunoprecipitations were assessed by Western blot analyses. Fig.1 B, bottom panel, shows that more than 80% of PKC λ, PKC α, or PKC δ were immunoprecipitated. Therefore, the co-immunoprecipitation of p90 with PKC λ is not due to a relatively larger proportion of PKC λ being immunoprecipitated compared with PKC α or PKC δ. The molecular mass and the gel migration pattern of the 90-kDa tyrosine-phosphorylated protein resembled that of FRS2, a protein that our laboratory is currently studying. To determine whether the p90 protein was FRS2, lysates from Swiss 3T3 cells that were untreated or stimulated with bFGF were subjected to immunoprecipitation of PKC λ. The immunoprecipitates were processed as described above. The blot was first probed with phosphotyrosine antibodies revealing the 90-kDa tyrosine-phosphorylated protein co-precipitating with PKC λ upon bFGF stimulation (Fig. 1 C, top panel). The blots were stripped and re-probed with A872, a polyclonal antibody raised against FRS2. As shown in Fig. 1 C, middle panel, FRS2 co-precipitated with PKC λ from lysates of bFGF-stimulated but not non-stimulated cells. It is noted that phosphotyrosine signal for p90 that co-immunoprecipitated with PKC λ (Fig. 1 C, top panel) is greater than that of FRS2 co-immunoprecipitated with PKC λ (Fig.1 C, middle panel). This is attributed to the observation that FRS2 is a multiply tyrosine-phosphorylated protein with at least 6 tyrosine phosphorylation sites. By comparing the amount of FRS2 in the" @default.
• W2018575189 created "2016-06-24" @default.
• W2018575189 creator A5014257263 @default.
• W2018575189 creator A5028573028 @default.
• W2018575189 creator A5055998574 @default.
• W2018575189 creator A5057776021 @default.
• W2018575189 creator A5085764509 @default.
• W2018575189 date "1999-07-01" @default.
• W2018575189 modified "2023-10-03" @default.
• W2018575189 title "Association of Atypical Protein Kinase C Isotypes with the Docker Protein FRS2 in Fibroblast Growth Factor Signaling" @default.
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