MARCKS phosphorylation by individual protein kinase C isozymes in insect Sf9 cells.
Relatively little is known about the substrate specificity of individual protein kinase C (PKC) isozymes, particularly with respect to physiologically relevant substrates. One class of prominent cellular substrates for PKC is represented by the myristoylated alanine-rich C kinase substrate, or MARCKS, protein. In the present study, we have used a baculovirus expression system to coexpress human MARCKS with eight different isozymes of PKC, to determine which isozymes are capable of phosphorylating MARCKS in intact cells. In Sf9 cells, coexpression of MARCKS with individual PKC isozymes led to the following increases in MARCKS phosphorylation: alpha, 3.6-fold; beta iota, 4.6-fold; beta mu, 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 ester led to a slight increase (20-30%) in MARCKS phosphorylation. PKC zeta did not phosphorylate MARCKS to any appreciable extent above control. In addition, in vitro kinetic analysis of PKC zeta showed that it has a 1000-fold lower affinity for a synthetic peptide comprising the MARCKS phosphorylation site domain compared to mixed conventional PKC isozymes from rat brain. These data indicate that MARCKS is a substrate in intact cells for at least seven isozymes of PKC: alpha; beta iota; beta mu; gamma; delta; epsilon; and eta. The isozyme PKC zeta does not appear to phosphorylate MARCKS in vivo or with significant affinity in vitro. Thus, PKC zeta, which is not activated by phorbol esters or diacylglycerol, also appears to behave differently with respect to this class of important cellular PKC substrates.
Cabell
C H
CH
Department of Medicine, Duke University Medical Center, Durham, NC, USA.
Verghese
G M
GM
Rankl
N B
NB
Burns
D J
DJ
Blackshear
P J
PJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D018411:Spodoptera; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619710:MARCKS protein, human; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C