title: Failure to myristoylate the alpha subunit of Gz is correlated with an inhibition of palmitoylation and membrane attachment, but has no affect on phosphorylation by protein kinase C.
abstract: The alpha subunit of the GTP-binding regulatory protein Gz has been shown to be a substrate for N-myristoylation, palmitoylation, and protein kinase C-catalyzed phosphorylation. In the present study, we used an NH2-terminal Gly-->Ala mutation to examine the relevance of myristoylation to phosphorylation and anchorage. 293 cells were transfected with cDNA encoding the normal or mutant form of alpha z. Normal alpha z was myristoylated and stably anchored, as assessed by biosynthetic labeling and sedimentation with membrane following cell lysis. The mutant form of alpha z was not myristoylated and was found predominantly (70-90%) in the cytosol. These distributions were corroborated by immunofluorescence microscopy. Thus, the NH2-terminal glycine of alpha z appears critical for both myristoylation and anchorage. The effects of the Gly-->Ala mutation, however, were not confined to these two events. Significantly, the mutation also prevented palmitoylation, which presumably occurs at Cys3 in the normal subunit. Therefore, it cannot be concluded that myristoylation alone is sufficient for anchorage. The protein kinase C-catalyzed phosphorylation of alpha z was demonstrated previously to occur near to the NH2 terminus, raising the possibility of an effect of phosphorylation on anchorage or, conversely, an effect of myristoylation and/or palmitoylation on phosphorylation. The phosphorylation induced in 293 cells with phorbol 12-myristate 13-acetate, however, had little impact on anchorage. Moreover, the mutant (and thus cytosolic) form of alpha z was as good a substrate for phosphorylation as the normal subunit. The fact that myristoylation occurs in cells containing alpha z normally, as well as the fact that phosphorylation does not influence anchorage, was confirmed with mouse anterior pituitary AtT-20 and rat basophilic RBL-2H3 cells. These results demonstrate: 1) that mutagenesis of the glycine needed for myristoylation of alpha z also inhibits palmitoylation, 2) that the resultant lack of myristoylation and/or palmitoylation does not support anchorage, and 3) that neither myristoylation, palmitoylation, nor anchorage is required for phosphorylation.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
authors.forename: H
authors.initials: H
authors.lastname: Hallak
authors.forename: L F
authors.initials: LF
authors.lastname: Brass
authors.forename: D R
authors.initials: DR
authors.lastname: Manning
mesh_terms: D000215:Acylation; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009226:Myristates / Q000378:metabolism*; D010168:Palmitates / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; D009226:Myristates; D010168:Palmitates; D011493:Protein Kinase C; D019204:GTP-Binding Proteins