MARCKS PHOSPHORYLATION BY INDIVIDUAL PROTEIN-KINASE-C ISOZYMES IN INSECT SF9 CELLS

Relatively little is known about the substrate specificity of individu al protein kinase C (PKC) isozymes, particularly with respect to physi ologically relevant substrates. One class of prominent cellular substr ates for PKC is represented by the myristoylated alanine-rich C kinase substrate, or MARCKS, protein. In the present study, we have used a b aculovirus expression system to coexpress human MARCKS with eight diff erent isozymes of PKC, to determine which isozymes are capable of phos phorylating MARCKS in intact cells. In Sf9 cells, coexpression of MARC KS with individual PKC isozymes led to the following increases in MARC KS phosphorylation: alpha, 3.6-fold; beta iota, 4.6-fold; beta iota io ta, 2.7-fold; gamma, 4.8-fold; delta, 3.0-fold; epsilon 4.3-fold; and eta, 4.9-fold. In most cases, stimulation of cells with a phorbol este r led to a slight increase (20-30%) in MARCKS phosphorylation. PKC zet a did not phosphorylate MARCKS to any appreciable extent above control . In addition, in vitro kinetic analysis of PKC zeta showed that it ha s a 1000-fold lower affinity for a synthetic peptide comprising the MA RCKS phosphorylation site domain compared to mixed conventional PKC is ozymes from rat brain. These data indicate that MARCKS is a substrate in intact cells for at least seven isozymes of PKC: alpha; beta iota b eta iota iota; gamma; delta; epsilon; and eta. The isozyme PKC zeta do es not appear to phosphorylate MARCKS in vivo or with significant affi nity in vitro. Thus, PKC zeta, which is not activated by phorbol ester s or diacylglycerol, also appears to behave differently with respect t o this class of important cellular PKC substrates.