Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.
We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.
Jefferson
A B
AB
Department of Pharmacology, Stanford University School of Medicine, California 94305-5322.
Travis
S M
SM
Schulman
H
H
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D008564:Membrane Potentials; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010449:Peptide Mapping; D010716:Phosphatidylinositols / Q000502:physiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity; D016704:Synapsins; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D002147:Calmodulin; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; D016704:Synapsins; D013973:Thyrotropin-Releasing Hormone; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium