Activation of multifunctional calcium/calmodullin dependent protein kinase and phosphorylation of MAP-2 in GH3 cells
The author utilized the pituitary-derived cell line, GH3, as a model system for studying the in situ regulation of multifunctional Ca{sup 2+}/calmodulin-dependent protein kinase (CaM kinase). The author partially purified a Ca{sup 2+}/ calmodulin-dependent protein kinase from GH3 cells and demonstrated that it is similar in biochemical properties to neuronal CaM kinase. Autophosphorylation at the autonomy site converts the kinase into a Ca{sup 2+}-independent enzyme. Regulation of CaM kinase in situ was examined by high K{sup +} depolarization of ({sup 32}P)Pi-labeled H3 cells followed by immunoprecipitation and trypic phosphopeptide mapping. The enzyme displayed a Ca{sup 2+} dependent increase in phosphorylation of the autonomy site. Accordingly, this led to a considerable increase in the Ca{sup 2+}-independent or autonomous activity of the enzyme. Thus, activation of CaM kinase by Ca{sup 2}/calmodulin and the subsequent formation of a Ca{sup 2+}-independent species, previously established in vitro, occur after Ca{sup 2+} influx in situ. In a parallel study the author tested whether microtubule-associated protein-2 (MAP-2), an in vitro substrate of CaM kinase, is phosphorylated by CaM kinase in GH3 cells. MAP-2 phosphorylation is enhanced by depolarization with high K{sup +} at sites characteristic of those recognized by CaM kinase and distinct from those phosphorylated by cAMP kinase or protein kinase C. Thyrotropin releasing hormone (TRH) increased Ca{sup 2+} via the phosphatidyl inositol signaling pathway but neither stimulated autophosphorylation of CaM kinase nor increased phosphorylation of the CaM kinase array of sites on MAP-2. TRH does increase MAP-2 phosphorylation but at sites which closely match those stimulated by phorbol esters that activate protein kinase C.
- Research Organization:
- Stanford Univ., CA (USA)
- OSTI ID:
- 6414654
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ANIMAL CELLS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL PATHWAYS
BODY
CALCIUM COMPOUNDS
CALMODULIN
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENDOCRINE GLANDS
ENZYME ACTIVITY
ENZYMES
FRACTIONATION
GLANDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PITUITARY GLAND
PROTEINS
RADIOISOTOPES
SEPARATION PROCESSES
TRACER TECHNIQUES
TRANSFERASES