title: Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes.
abstract: 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 (approximately 80% membrane, approximately 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
authors.forename: G
authors.initials: G
authors.lastname: James
authors.forename: E N
authors.initials: EN
authors.lastname: Olson
mesh_terms: D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009227:Myristic Acids; D010710:Phosphates; D010750:Phosphoproteins; D011506:Proteins; D019814:Myristic Acid; D015240:Phorbol 12,13-Dibutyrate; D003513:Cycloheximide; D011493:Protein Kinase C