Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC₃H1 myocytes

Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC₃H1 myocytes and have examined the subcellular distribution of the [³H]myristate and ³²P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC₃H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [³H]myristate. Incorporation of [³H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [³H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (~80% membrane, ~20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC₃H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.