437 more than two samples. FAK stimulated cell migration as effectively as wild-type Pyk2; however, substitution of the N-terminal domain of Pyk2 with that of FAK inhibited cell migration, substantiating that the N-terminal domain of Pyk2 was required to stimulate migration. Silencing of Pyk2 expression by RNA interference significantly inhibited glioma migration. Cell migration was restored on reexpression of Pyk2, but expression of FAK in Pyk2 knockdown cells failed to restore migration. We conclude that Pyk2 plays a central role in the migratory behavior of glioblastomas. and [16C18]. Furthermore, increased FAK activity has been correlated with increased cell proliferation and cell cycle progression, which play integral roles in tumor progression [19C21]. Indeed, overexpression of FAK promoted malignant astrocytoma cell proliferation [22]. In contrast, it has been reported that although FAK was localized to the membrane of nonmigratory astrocytoma cells, it was largely absent in migrating cells [23]. High levels of endogenous FAK also have been correlated with reduced migratory rates of cultured glioma cells [24]. Furthermore, dephosphorylation and downregulation of FAK activity correlated positively with EGF-induced tumor cell invasion [25]. The related focal adhesion kinase Pyk2 has a conserved domain structure and significant sequence identity to FAK [26,27]. Although Pyk2 can interact with many of the same proteins as FAK, it has a different primary means of activation and a more limited tissue distribution. Notably, Pyk2 is highly expressed in the central nervous system [28]. In contrast to the more rigorously studied FAK, the cellular functions of Pyk2 continue to be defined. Although FAK activity has been readily linked to cell motility, a role for Pyk2 Kaempferol-3-O-glucorhamnoside in cell migration has only recently begun to be appreciated. In cell types that naturally express Pyk2 but little or no FAK, such as NK cells, monocytes, or osteoclasts, a critical role for Pyk2 in the regulation of cytoskeletal organization and cell motility has been established [29C32]. Therefore, in cells such as gliomas that express both kinases, the temporal balance between Pyk2 and FAK activity may ultimately regulate cell growth and cell migration. The recent demonstration [33] of a clear correlation between expression of FAK and Pyk2 and increasing Rabbit polyclonal to cytochromeb malignancy suggests that the differential activity of Kaempferol-3-O-glucorhamnoside these two kinases may be particularly relevant to the regulation of the proliferative or migratory behavior of these cells. The current study investigated the functional contributions of FAK and Pyk2 in determining glioma cell migration and proliferation. Expression of Pyk2 stimulated glioma cell migration in a dose-dependent manner. Autophosphorylation of Pyk2 Y402 was necessary but Kaempferol-3-O-glucorhamnoside insufficient for this phenotypic effect. The N-terminal FERM domain of Pyk2 was required to stimulate migration and was capable of inhibiting migration when expressed as an autonomous fragment. In contrast, expression of FAK inhibited glioma cell migration and stimulated cell cycle progression in a dose-dependent manner. Knockdown of Pyk2 expression by RNAi inhibited glioma cell migration and in organotypic brain slices. Reconstitution of Pyk2 expression in these cells, but not FAK expression, restored glioma cell migration. These data further indicate that Pyk2 and FAK function in the temporal development of the proliferative or migratory behavior of glioma Kaempferol-3-O-glucorhamnoside cells and may be useful therapeutic targets in malignant glioblastoma. Materials and Methods Antibodies Affinity-purified polyclonal antibodies to the C-terminal portion of FAK were obtained from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibody to the C-terminal portion of Pyk2 was obtained from Transduction Laboratories (Lexington, KY). Anti-FLAG M2 monoclonal antibody was obtained from Sigma (St. Louis, MO). Affinity-purified antibody to the HA epitope tag was obtained from Upstate Biotechnology. Adenoviral Expression Constructs Recombinant adenoviruses encoding HA epitope-tagged wild-type FAK was previously described [24]. To generate FLAG epitope-tagged wild-type Pyk2, the coding sequence or Pyk2 was cloned in-frame downstream of a 3x FLAG epitope in pcDNA3. The Pyk2 phosphorylation-deficient was generated using the Quickchange site directed mutagenesis system (Stratagene, La Jolla, CA). To generate the Pyk2 N-terminal deletion mutant Pyk2376, a fragment encoding Pyk2 residue S376-E1009 was amplified by polymerase chain reaction (PCR) Kaempferol-3-O-glucorhamnoside and cloned in-frame downstream of the 3x FLAG epitope in pcDNA3. The FLAG epitope-tagged chimeric proteins designated Pyk/FAK and FAK/Pyk were generated by splice overlap extension PCR [34]. Pyk/FAK contains Pyk2 N-terminal residue M1-D665 fused to FAK residue P663-H1052. FAK/Pyk.
437 more than two samples