Analysis of the calcium-dependent regulation of proline-rich tyrosine kinase 2 by gonadotropin-releasing hormone.
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.
Xie
Jianjun
J
Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853, USA.
Allen
Krystal H
KH
Marguet
Amelia
A
Berghorn
Kathie A
KA
Bliss
Stuart P
SP
Navratil
Amy M
AM
Guan
Jun Lin
JL
Roberson
Mark S
MS
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology
D015220:Calcium Channels; D002147:Calmodulin; D034741:RNA, Small Interfering; D007987:Gonadotropin-Releasing Hormone; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium