Phosphorylation by protein kinase C of a 20-kDa cytoskeletal polypeptide enhances its susceptibility to digestion by calpain.

Abstract

Incubation of the cytoskeletal fraction from human neutrophils with the proteolytically activated form of protein kinase C results in the phosphorylation of several components, including a 20-kDa polypeptide, probably consisting of myosin light chains. The 20-kDa polypeptide is also specifically phosphorylated by activated protein kinase C in a solubilized 20-kDa/80-kDa complex that was obtained after sonication of the insoluble cytoskeletal fraction. Phosphorylation of this polypeptide, in either the insoluble cytoskeletal fraction or the soluble 20-kDa/80-kDa complex, greatly enhances its susceptibility to digestion by the Ca2+-requiring proteinase (calpain, EC 3.4.22.17) of human neutrophils. Thus, signals that activate calpain by mobilizing intracellular calcium would lead to proteolytic activation of protein kinase C, phosphorylation of cytoskeletal proteins, and remodeling of the cytoskeleton by proteolysis of at least one cytoskeletal component.

An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

“…The values in parentheses represent the percentage of recovery of the activator and inhibitor activities, based on their content in the crude extracts calculated from an experiment similar to that shown in Fig. 1 (16,27), presumably in the presence of less than micromolar concentrations of Ca2W, prompted the present search for a cytosolic activator and particularly for an activator associated with the cytoskeletal fraction. The cytoskeletal protein described in the present work fulfills the requirements for a physiological activator; it increases the affinity of neutrophil calpain for Ca2' by >100-fold, and in the presence of the activator significant calpain activity is expressed in the presence of 0.25 ,uM Ca2+ (see Fig.…”

“…The presence of a soluble calpastatin that fully antagonizes the effects of the activator, but only at low concentrations of Ca2+, raises interesting questions concerning the physiological role of these modulators of calpain activity. Calpastatin would protect cytosolic proteins from proteolysis, even in the presence of elevated cytosolic Ca2" Association of calpain with the plasma membrane or with the cytoskeletal fraction would result in digestion of specific membrane or cytoskeletal proteins-e.g., protein kinase C in the case of membranes (18) or a phosphorylated polypeptide associated with the cytoskeletal fractions (16,27). Calpain and the proteolytically modified form of protein kinase C are emerging as important components of the signal transduction mechanism of human neutrophils.…”

Post‐translational Modification of Peptide Messengers in the Gut

“…In the light of the importance of phosphorylation-dephosphorylation reactions in controlling regulatory protein activity, it is possible that phosphorylation identifies a site as important for subsequent cleavage. There are several examples where phosphorylation identifies proteins for subsequent intracellular degradation (Mazon, Gancedo & Gancedo, 1982;Pontremoli, Melloni, Michetti, Sparatore, Salamino, Sacco & Horecker, 1987). Potential phosphorylation sites similar to that in the C-terminal region of progastrin also occur adjacent to important cleavage sites in other peptide precursors (Fig.…”

Phosphorylation and Proteolytic Degradation of Neurofilaments

“…Phosphorylation of proteins can either enhance or decrease their sensitivities to proteases [2][3][4][5]. We have shown in this pa per that phosphorylation of NFs by PK-C or PK-P decreases their susceptibility to pro teolytic digestion, whereas phosphorylation by PK-A or p34 kinase did not (Fig.…”

“…2], For example, phosphorylation by protein kinase C (PK-C) of a cytoskeletal protein with an Mr of 20,000 enhances its susceptibility to digestion by calpain at low Ca2+ concentrations [3]. In yeast, phosphory lation of fructose-1,6-bisphosphatase mark edly increased the proteolytic degradation of this enzyme [4].…”