Protein kinase C-induced phosphorylation of MARCKS and GAP-43 in SK-N-SH human neuroblastoma cells.

Abstract

Protein kinase C (PKC)-induced phosphorylation of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the 43 kDa growth-associated protein (GAP-43) decreases the affinity of these membrane-associated proteins for the Ca$\sp{2+}$ binding protein, calmodulin (CaM). These proteins may sequester CaM in unstimulated cell membranes and upon phosphorylation release CaM into the cytosol where CaM can activate enzymes or bind cytoskeletal elements. This thesis examined PKC-mediated phosphorylation of MARCKS and GAP-43 in SK-N-SH human neuroblastoma cells. PKC phosphorylated MARCKS in response to the muscarinic agonist carbachol, a PKC-activating phorbol ester (TPA) and the Ca$\sp{2+}$ ionophore ionomycin. GAP-43 was phosphorylated in response to TPA only. Only PKC $\alpha$ translocated to cell membranes in response to ionomycin, while only PKC $\varepsilon$ translocated in response to TPA. In permeabilized cells, GAP-43 cross-linked to exogenously added CaM but binding was unaffected by drug treatment. No cross-linking of MARCKS or CaM was demonstrated in the cells. These results suggest GAP-43 and MARCKS are not involved in CaM sequestration and redistribution. Long-term treatment with TPA, which down-regulates PKC isozymes, induced the constitutive phosphorylation of MARCKS at 17 and 72 h and of GAP-43 at 17 h of treatment. GAP-43 content increased 2.3-fold at 17 h and 3.7-fold at 72 h of TPA treatment but MARCKS content was unchanged. The ratio of phosphorylation to GAP-43 content was increased at 17 h but returned to basal levels by 72 h of TPA treatment. This suggests down-regulation of PKC isozymes that phosphorylate GAP-43 or compartmentalization of PKC and/or the newly synthesized GAP-43. Short- and long-term TPA treatment induced phosphorylation of MARCKS and GAP-43 at the same sites. PKC $\alpha$ and $\varepsilon$ were down-regulated mainly in the cytosol while PKC $\beta$ and $\zeta$ were unaffected. PKC activity was nearly abolished in the cytosol while significant activity remained in the membranes. The data from this thesis suggest that the phosphorylation states of MARCKS and GAP-43 are differentially regulated by PKC which may prove important in how CaM is regulated by phosphorylation of these and other CaM-binding proteins.