Endogenous FGF1-induced activation and synthesis of extracellular signal-regulated kinase 2 reduce cell apoptosis in retinal-pigmented epithelial cells

Retinal-pigmented epithelial (RPE) cell survival is critical to the maintenance of the function of the neural retinal and in the development of various retina degenerations. We investigated molecular mechanisms involved in this function by assessing apoptosis in RPE cells following serum deprivation. Apoptosis induced by serum withdrawal is lower in aged RPE cells because of higher endogenous acidic fibroblast growth factor (FGF1) synthesis and secretion. These experiments examined several aspects of FGF signaling and the contribution of endogenous FGF1 to activation of the extracellular signal-regulated kinase 2 (ERK2). In aged RPE cells, FGFR1 was rapidly activated, and its autophosphorylation followed the kinetics of endogenous FGF1 secretion, before the onset of apoptosis. ERK2 phosphorylation, activity, and de novo synthesis increased at the same time. In marked contrast, no de novo JNK1 synthesis was observed. MEK1 inhibition resulted in lower levels of ERK2 activation and synthesis and higher levels of apoptosis. Treatment with neutralizing anti-FGF1 or blocking anti-FGFR1 antibodies mimics these effects. Thus, this study strongly suggests that the survival-increasing effect of FGF1 in aged RPE cells is because of an autocrine/paracrine loop in which the ERK2 cascade plays a pivotal role.     

Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth and activating mutations in Fgfr3 cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, we targeted the expression of an activated FGFR3 to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. We found that FGFR3 inhibited endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulated the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium.     

Common and specific determinants for fibroblast growth factors in the ectodomain of the receptor kinase complex

The assembly and activation of oligomeric complexes of FGF, the transmembrane receptor kinase (FGFR), and heparan sulfate transmit intracellular signals regulating growth and function of cells. An understanding of the structural relationships between the three subunits and their redundancy and specificity is essential for understanding the ubiquitous FGF signaling system in health and disease. Previously, we reported that a primary heparin or heparan sulfate binding site resides in a distinct sequence in immunoglobulin (Ig)-like module II of the three modules of FGFR. Here we report that in the absence of flanking sequences, isolated Ig module II of FGFR1 supports the binding of FGF-1, FGF-2, and FGF-7 in respective order of affinity. None of the three FGFs detectably bind Ig module I or the IIIb and IIIc splice variants of Ig module III in the absence of flanking sequences. Ig module I and the C-terminus of Ig module III are dispensable for high-affinity binding of FGF-1, FGF-2, and FGF-7. Alterations in highly conserved Ig module II in the heparin binding domain and substitution of individual sequence domains spanning the entire sequence of Ig module II with those from Ig module I obliterated FGF binding. Addition of a specific number of FGFR sequences to the C-terminus of Ig module II resulted in a gain in affinity for FGF-7. Several site-specific alterations in the C-terminus of full-length FGFR1IIIc, an isoform that otherwise absolutely rejects FGF-7, resulted in gain of FGF-7 binding. These results suggest that a complex of Ig module II and heparan sulfate is the base common active core of the FGFR ectodomain and that flanking structural domains modify FGF affinity and determine specificity.     

Calmodulin inhibitor W13 induces sustained activation of ERK2 and expression of p21(cip1)

One of the major signaling pathways by which extracellular signals induce cell proliferation and differentiation involves the activation of extracellular signal-regulated kinases (ERKs). Because calmodulin is essential for quiescent cells to enter cell cycle, the role of calmodulin on ERK2 activation was studied in cultured fibroblasts. Serum, phorbol esters, or active Ras induced ERK2 activation in NIH 3T3 fibroblasts. This activation was not inhibited by calmodulin blockade. Surprisingly, inhibition of calmodulin prior to fetal bovine serum addition prolonged activation of ERK2. Furthermore, inactivation of calmodulin in serum-starved cells induced ERK2 phosphorylation that was dependent on MAP kinase kinase (MEK). Inactivation of calmodulin in serum-starved cells also induced activation of Ras, Raf, and MEK. On the contrary, tyrosine phosphorylation of tyrosine kinase receptors was not observed. These results indicate that calmodulin inhibits ERK2 activation pathway at the level of Ras. Calmodulin inhibition induced overexpression of p21(cip1) which was dependent on MEK activity. We propose that inhibition of Ras by calmodulin prevents the activation of ERK2 at low serum concentration. Thus, entering into the cell cycle after serum addition would imply the overcoming of the inhibitory effect of calmodulin and consequently ERK2 activation. Furthermore, down-regulation of Ras by calmodulin may be also important to determine the duration of ERK2 activation and to prevent a high p21(cip1) expression that would lead to an inhibition of cell proliferation.     

Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.     

Identification of the cytoplasmic regions of fibroblast growth factor (FGF) receptor 1 which play important roles in induction of neurite outgrowth in PC12 cells by FGF-1

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.     

Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation

Insulin-like growth factor-I (IGF-I) induces neuronal differentiation in vitro. In the present study, we examined the signaling pathway underlying IGF-I-mediated neurite outgrowth. In SH-SY5Y human neuroblastoma cells, treatment with IGF-I induced concentration- and time-dependent tyrosine phosphorylation of the type I IGF receptor (IGF-IR) and extracellular signal-regulated protein kinases (ERK) 1 and 2. These effects of IGF-I were blocked by a neutralizing antibody against IGF-IR. Whereas IGF-IR phosphorylation was observed within 1 min, maximal phosphorylation of ERKs was not reached for 30 min. Both IGF-IR and ERK phosphorylation were maintained for at least 24 h. Also, the concentration dependence of IGF-I-stimulated IGF-IR and ERK tyrosine phosphorylation paralleled that of IGF-I-mediated neurite outgrowth. We further examined the role of mitogen-activated protein kinase activation in IGF-I-stimulated neuronal differentiation using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059. Whereas PD98059 had no effect on IGF-IR phosphorylation, PD98059 reduced IGF-I-mediated ERK tyrosine phosphorylation and ERK phosphorylation of the substrate Elk-1. PD98059 also produced a parallel reduction of IGF-I-stimulated neurite outgrowth. Finally, consistent with its ability to block neuronal differentiation, PD98059 inhibited IGF-I-dependent changes of GAP-43 and c-myc gene expression. Together these results suggest that activation of ERKs is essential for IGF-I-stimulated neuronal differentiation.     

Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1

BACKGROUND:. Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the non-receptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk. RESULTS:. Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin alphaIIbbeta3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Syk-dependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with alphaIIbbeta3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization. CONCLUSIONS:. Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3'-kinase-regulated kinase Akt.     

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.     

Hypoxic activation of nuclear factor-kappa B is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2)

We have previously shown that hypoxia causes the activation of nuclear factor-kappa B (NF-kappa B), and the phosphorylation of its inhibitory subunit, I kappa B alpha, on tyrosine residues. With the use of dominant negative mutants of Ha-Ras and Raf-1, we investigated some of the early signaling events leading to the activation of NF-kappa B by hypoxia. Both dominant negative alleles of Ha-Ras and Raf-1 inhibited NF-kappa B induction by hypoxia, suggesting that the hypoxia-induced pathway of NF-kappa B induction is dependent on Ras and Raf-1 kinase activity. Furthermore, although conditions of low oxygen can also activate mitogen-activated protein kinases (ERK1 and ERK2), these kinases do not appear to be involved in regulating NF-kappa B by low oxygen conditions, as dominant negative mutants of mitogen-activated protein kinase do not inhibit NF-kappa B activation by hypoxia. Since Ras and Raf-1 have been previously shown to work downstream from membrane-associated tyrosine kinases such as Src, we determined if the Src membrane-associated kinase was also activated by low oxygen conditions. We detected an increase in Src proto-oncogene activity within 15-30 min of cellular exposure to hypoxia. We postulate that Src activation by hypoxia may be one of the earliest events that precedes Ras activation in the signaling cascade which ultimately leads to the phosphorylation and dissociation of the inhibitory subunit of NF-kappa B, I kappa B alpha.     

Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphorylation

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac.     

Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells. Essential role for a herbimycin-sensitive kinase

Fluid shear stress modulates vascular function and structure by stimulating mechanosensitive endothelial cell signal events. Cell adhesion, mediated by integrin-matrix interactions, also regulates intracellular signaling by mechanosensitive events. To gain insight into the role of integrin-matrix interactions, we compared tyrosine phosphorylation and extracellular signal-regulated kinase (ERK1/2) activation in adhesion- and shear stress-stimulated human umbilical vein endothelial cells (HUVEC). Adhesion of HUVEC to fibronectin, but not to poly-L-lysine, rapidly activated ERK1/2. Fluid shear stress (12 dyn/cm2) enhanced ERK1/2 activation stimulated by adhesion, suggesting the presence of a separate pathway. Two differences in signal transduction were identified: focal adhesion kinase phosphorylation was increased rapidly by adhesion but not by shear stress; and ERK1/2 activation in response to adhesion was inhibited to a significantly greater extent when actin filaments were disrupted by cytochalasin D. Two similarities in activation of ERK1/2 were observed: protein kinase C (PKC) activity was necessary as shown by complete inhibition when PKC was downregulated; and an herbimycin-sensitive (genistein- and tyrphostin-insensitive) tyrosine kinase was required. c-Src was identified as a candidate tyrosine kinase as it was activated by both shear stress and adhesion. These findings suggest that adhesion and shear stress activate ERK1/2 via a shared pathway that involves an herbimycin-sensitive tyrosine kinase and PKC. In addition, shear stress activates ERK1/2 through another pathway that is partially independent of cytoskeletal integrity.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

Role of cyclooxygenase-2 for fluid secretion by the inflamed gallbladder mucosa

Heterotrimeric GTP-binding protein (G-protein)-coupled receptors are able to induce a variety of responses including cell proliferation, differentiation, and activation of several intracellular kinase cascades. Prominent among these kinases are the activation of mitogen-activated protein (MAP) kinase, including the extracellular signal-regulated kinases (ERKs), ERK1 and ERK2 (p44mapk and p42mapk, respectively); stress-activated protein kinases (SAPKs/JNKs); and p38 kinase. These receptors signal through G-proteins. Recent data have shown that the activation of mitogen-activated protein/ERK kinase induced by G-protein-coupled receptors is mediated by both Galpha and Gbetagamma subunits involving a common signaling pathway with receptor-tyrosine-kinases. Gbetagamma-mediated mitogen-activated protein kinase activation is mediated by activation of phosphoinositide 3-kinase, followed by a tyrosine phosphorylation event, and proceeds in a sequence of events that involve functional association among the adaptor proteins Shc, Grb2, and Sos. SAPKs/JNKs and p38 are able to be activated by Gbetagamma proteins in a pathway involving Rho family proteins including RhoA, Rac1, and Cdc42.     

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 intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy 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 (FGFR1-4F) 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 pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.