Overexpression of Ca²⁺/Calmodulin-Dependent Protein Kinase II in PC12 Cells Alters Cell Growth, Morphology, and Nerve Growth Factor-Induced Differentiation

Abstract

To examine the role of Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) in cell differentiation and neuronal functions, stable transformants of PC12 cells were established that expressed levels of the ␣-subunit of CaMKII (␣CaMKII) equivalent to mammalian neurons. The expression of the transfected ␣CaMKII gene or the endogenous ␤CaMKII gene was monitored by RNase protection assays, and ␣CaMKII protein expression was determined by Western blots. Several PC12-derived clones expressed amounts of ␣CaMKII mRNA and ␣CaMKII protein similar to that of hippocampal tissues and several orders of magnitude greater than untransfected PC12 cells. CaMKII catalytic activity was four times higher in extracts from ␣CaMKII-overexpressing compared with untransfected PC12 cells. All clones overexpressing ␣CaMKII displayed altered cellular growth and adhesion properties including increased cell-to-substrate adhesion, decreased cell-to-cell adhesion, enhanced contact inhibition, and prolonged survival at confluency. Furthermore, the ␣CaMKII activity in overexpressing PC12 cells inhibited neurite elongation during NGF-induced differentiation. Inhibition of CaMKII activity in vivo with KN-62 caused the morphological phenotypes of ␣CaMKII-overexpressing cells to partially revert to that of untransfected PC12 cells. These results show that ␣CaMKII catalytic activity affects growth, morphology, and NGF-induced differentiation of PC12 cells.

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates Raf-1 at serine 338 and mediates Ras-stimulated Raf-1 activation

“…An excess of enzyme is sufficient to activate the CaMKII complex also in the absence of a stimulus. 35 After 48 h starvation, cell lysate (CL) or immunoprecipitated CaMKII were submitted to SDS-pAGe, blotted onto nitrocellulose membrane and Raf-1 or B-Raf were detected by specific antibodies.…”

Identification and Expression of δ-Isoforms of the Multifunctional Ca 2+ /Calmodulin-Dependent Protein Kinase in Failing and Nonfailing Human Myocardium

“…4,5 Alternative splicing of the primary transcripts encoded by 4 genes (␣, ␤, ␥, and ␦) results in the generation of different CaMKII variants. 6,7 Members of the ␣-and ␤-classes are expressed mainly in neuronal tissue, whereas ␥-and ␦-CaMKII isoforms are abundantly expressed in all tissues.…”

Multiple Distinct Signal Pathways, Including an Autocrine Neurotrophic Mechanism, Contribute to the Survival-Promoting Effect of Depolarization on Spiral Ganglion NeuronsIn Vitro

“…CaMK (Hack et al, 1993;Massé and Kelly, 1997;Vaillant et al, 1999) and CaMK kinase, acting via PKB (Yano et al, 1998), have been implicated in depolarization-dependent survival. CaMKs are activated by depolarization in SGNs, inhibition of CaMK activity compromises depolarization-dependent survival, and CaMK activity is sufficient to support SGN survival in the absence of exogenous survival-promoting factors (our unpublished observations).…”