The Cytoplasmic Tyrosine Kinase Pyk2 as a Novel Effector of Fibroblast Growth Factor Receptor 3 Activation

Abstract

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr 402 , the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr 402 , indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr 402 of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.Receptor tyrosine kinases comprise nearly two dozen different families of homologous proteins in humans, with at least 40 distinct members, and are responsible for the integration of many different signals that affect development, proliferation, and homeostasis. In the fibroblast growth factor receptor (FGFR) 1 family, four homologous receptors have been identified, designated FGFR1, FGFR2, FGFR3, and FGFR4. The basic structure of FGFRs consists of three extracellular Ig-like domains stabilized by characteristic disulfide bonds, a single membrane-spanning segment, and an intracellular split tyrosine kinase domain (1, 2). Fibroblast growth factors, a large family of at least 20 growth factors, act in concert with heparan sulfate proteoglycans as high affinity agonists that induce FGFR dimerization and transphosphorylation (3-6). Activation of FGFRs controls an array of biological processes, including cell growth, differentiation, migration, wound healing, and angiogenesis. FGFRs and their ligands play a major role in autocrine and paracrine signaling loops that have been implicated in a number of malignancies, including cancers of the stomach, breast, thyroid, prostate, and pancreas and also some leukemias (7,8).A frequent translocation observed in multiple myeloma, t(4; 14)(p16.3;q32.3), involves the FGFR3 gene and results in increased expre...

Type I Collagen Limits VEGFR-2 Signaling by a SHP2 Protein-Tyrosine Phosphatase–Dependent Mechanism 1

“…This residue seems important in internalisation process as demonstrated by studying truncated receptors lacking Tyr-1173 and Tyr-1212. 61 This process occurs in ECs growing on native ECM, as demonstrated by the data showing that this phosphatase is also activated by VEGF-A 165 in ECs adherent to this ECM (Figures 2A, 2B, and 3) as well as in cells on collagen I. Here, we did not address the behavior of the internalized VEGFR-2 in EC adhering on collagen I. VEGFR-2 could either be accumulated in the endosomal compartment and degraded or recycled to the membrane, thus allowing a different degree of EC activation or a modified spatial regulation of the signal.…”

Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

“…It has also been shown that the FGFR3 juxtamembrane domain interacts with Pyk2, a focal adhesion kinase, whose activity is required for STAT5 phophorylation. However, FGFR3 (TDII) can override the requirement of Pyk2 for the activation of STAT5 [136]. STAT5, which is known to induce cyclin D1 expression, has been shown to be involved in cell proliferation and may represent a good candidate for downstream signalling in FGFR3-positive cancers [137].…”

K644E/M FGFR3 Mutants Activate Erk1/2 from the Endoplasmic Reticulum through FRS2α and PLCγ-independent Pathways

“…Since the cytoplasmic tyrosine kinase Pyk2 has been recently indicated as a novel effector of FGFR3 activation, 27 we investigated whether Pyk2 could play any role in the signaling pathway triggered by the SADDAN and TDII mutants. For this purpose, cell lysates from transiently transfected HEK293 cells were compared with cell lysates from the stable cell lines.…”