Katherine A. Albert

Insulin increases membrane protein kinase C activity in rat diaphragm

Calcium/phospholipid‐dependent protein kinase activity (protein kinase C) was identified in rat diaphragm membrane and cytosol fractions by means of in vitro phosphorylation either of histones or of a specific 87 kDa protein substrate, combined with phosphopeptide‐mapping techniques. Both insulin and tumor‐promoting phorbol ester treatment of the diaphragm preparations led to increased protein kinase C activity in the membrane fractions. In contrast to the phorbol ester, however, insulin did not induce a concomitant decrease in cytosolic activity, indicating that translocation of the enzyme had not taken place. Thus, insulin appears to increase specifically membrane protein kinase C activity in rat skeletal muscle, possibly through a mechanism not identical to that induced by phorbol esters.

Phosphorylation of multiple sites in a 15 000 dalton proteolipid from rat skeletal muscle sarcolemma, catalyzed by adenosine 3′,5′-monophosphate-dependent and calcium / phospholipid-dependent protein kinases

This study reports a partial characterization of a 15,000 dalton (15 kDa) proteolipid present in rat skeletal muscle sarcolemma. The proteolipid is phosphorylated by both cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases, displays an isoelectric point (pI) of 5.9, and can be extracted from sarcolemma by acidified chloroform/methanol (2:1) or non-ionic detergents. Phosphoamino acid analysis and tryptic fingerprinting of the phosphorylated proteolipid indicate that both cyclic AMP- and calcium/phospholipid-dependent protein kinases predominantly phosphorylate serine residue(s) on a single tryptic peptide. Additivity experiments and thermolytic fingerprinting demonstrate a minimum of two distinct phosphorylation sites on the proteolipid, the phosphorylation of which is independently catalyzed by cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases in vitro. This sarcolemma proteolipid, which appears to be identified to a sarcolemma protein previously reported to be phosphorylated upon addition of insulin in a GTP-dependent manner (Walaas, O., Walaas, E., Rye-Alertsen, A. and Horn, R.S. (1979) Mol. Cell. Endocrinol. 16, 45-55), therefore represents a possible membrane target for those neuronal and hormonal stimuli which can regulate cyclic AMP-dependent or calcium/phospholipid-dependent protein kinase activities in skeletal muscle.

Calcium/Diacylglycerol‐Dependent Protein Kinase and Its Major 87‐Kilodalton Protein Substrate Are Differentially Distributed in Rat Basal Ganglia

Abstract: When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol‐depenpent protein kinase (protein kinase C) and an 87‐kiIodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chen|iical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87‐kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal gaijglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87‐kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87‐kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.