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Mol Endocrinol ; 22(10): 2322-35, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18635666

ABSTRACT

Calcium influx through L-type voltage-gated calcium channels (VGCC) is required for ERK activation induced by GnRH in pituitary gonadotropes. The current studies investigate VGCC-sensitive catalytic activities that may lie upstream of ERKs within the GnRH signaling network. Ion exchange fractionation of alphaT3-1 cell lysates subjected to anti-phosphotyrosine Western blot analysis revealed a nifedipine-sensitive activity that colocalized with proline-rich tyrosine kinase (Pyk) 2 immunoreactivity. Phosphorylated Pyk2 was present in alphaT3-1 cells after GnRH agonist administration for a time course that lasted up to 4 h. Pyk2 phosphorylation was also evident in gonadotropes in vivo after administration of a bolus of GnRH. Knockdown of Pyk2 using specific small interfering RNAs revealed that Pyk2 contributed to modulation of GnRH-induced ERK but not c-Jun N-terminal kinase activation. Using pharmacological approaches, calmodulin (Cam) was also demonstrated to be required for the phosphorylation of Pyk2. Pyk2 was shown to bind specifically to a Cam agarose affinity column in a calcium-dependent manner, suggesting Cam and Pyk2 are capable of forming a complex. Specific mutation of a putative Cam binding motif within the catalytic domain of Pyk2 blocked association with Cam and uncoupled Pyk2's ability to activate ERK-dependent gene transcription. Thus, GnRH induces Pyk2 tyrosine phosphorylation dependent upon calcium flux within gonadotropes. Furthermore, association of Pyk2 and Cam may be required to mediate the effects of calcium on Pyk2 phosphorylation and subsequent activation of ERKs by GnRH.


Subject(s)
Calcium/metabolism , Focal Adhesion Kinase 2/metabolism , Gonadotropin-Releasing Hormone/metabolism , Amino Acid Sequence , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calmodulin/metabolism , Catalytic Domain , Cell Line , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Focal Adhesion Kinase 2/genetics , Focal Adhesion Protein-Tyrosine Kinases/chemistry , Focal Adhesion Protein-Tyrosine Kinases/genetics , Humans , Mice , Molecular Sequence Data , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Sequence Alignment , Signal Transduction/physiology
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