Identiﬁcation of Six Novel Autophosphorylation Sites on Fibroblast Growth Factor Receptor 1 and Elucidation of Their Importance in Receptor Activation and Signal Transduction

Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intracellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase C (cid:103) and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identiﬁcation of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654, and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 are able to phosphorylate Shc and an unidentiﬁed Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and may therefore be the key link between FGFR1 and the Ras signaling pathway, mito- genesis, and neuronal differentiation. Our results indicate have found that with a 5 triple-mutant Y463/583/585F on the double mutant Y583/ 585F Y463/583/585F triple mutant ssm13 was then used to generate the Y463/583/585/730F (FGFR1-4F) mutant FGF Y583/ 585/766F triple-mutant receptor was generated on the mutant Y766F as the triple mutant Y583/585/766F used to generate the Y463/ 583/585/766F mutant Y463/583/585/766F was used as the template to the Y463/583/585/730/766F (FGFR1-5F) mutant FGF re- ceptor. mutated m13MP19 Klenow subcloned pMJ30 myoblasts, endogenous FGF transfected of and of or in pMJ30-expressing tryptic digestion 32 P-labeled same gra- dient and 32 P-labeled were analyzed by a pulsed on-line throughout these studies: anti-FGFR1 (29); anti-PLC (cid:103) 1 (45); anti-Sos1 (44), and anti-Grb2 and anti-Shc (2). In the in vivo labeling experiments we used an anti-Flg3B antiserum. These antibodies were raised against the kinase insert region of FGFR1 (residues 580 to 596) (52). In vitro kinase assay. Cells expressing wild-type and FGFR1-Y653F, FGFR1- Y654F, and FGFR1-Y653/654F mutant receptors were stimulated with aFGF (100 ng/ml) for 5 min at 37 (cid:56) C, lysed, and immunoprecipitated with anti-FGFR1 receptor antibodies. The immunoprecipitates were subjected to an in vitro kinase assay (50 (cid:109) l) in the presence of [ (cid:103) - 32 P]ATP (5 (cid:109) M), 10 mM Mg 2 Cl, and an exogenous substrate, either 5 mM angiotensin or a fusion protein containing the carboxy-terminal tail of FGFR1 fused to glutathione S -transferase (GST- FGFR1-CT) (10 (cid:109) g per reaction), for 5 min. Sample buffer was added, and the reaction products were analyzed by SDS-PAGE and autoradiography. Angioten- sin was separated from [ (cid:103) - 32 P]ATP by using a gradient gel of 5 to 15% (15 ml) that was layered on the top of a previously poured 20% gel (10 ml). PI hydrolysis. L-6 myoblasts expressing wild-type or mutant receptors were labeled with [ 3 H]myoinositol (2 (cid:109) Ci/ml) in DMEM containing 0.5% FBS for 24 h and incubated in DMEM containing 20 mM LiCl for 20 min before addition of aFGF (100 ng/ml) for an additional 30 min at 37 (cid:56) C. Cells perchloric acid and inositol phosphate formation