Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12-myristate 13-acetate.

Abstract

Stimulation of intact human neutrophils with phorbol 12-myristate 13-acetate results in the selective phosphorylation of two cytoskeletal protein components with molecular masses of 20 and 48 kDa. After phosphorylation the 48-kDa protein is no longer recovered as a component of the cytoskeletal fraction but is present as a fully soluble phosphoprotein. Phosphorylation of the 20-kDa protein (probably myosin light chains) signals a proteolytic conversion, catalyzed by calpain, to a smaller species having a molecular mass of approximately 15 kDa. Phosphorylation of both the 48-and 20-kDa proteins is related to the conversion of protein kinase C, also catalyzed by calpain, to the soluble fully active form. Leupeptin, an inhibitor of calpain, blocks both the phosphorylation of the target proteins and the proteolytic modification of the 20-kDa polypeptide. Thus, phosphorylation of cytoskeletal proteins and signal-directed proteolysis appear to be related processes that follow stimulation of human neutrophils by phorbol esters. The resulting changes in cytoskeletal organization may be involved in the expression of some neutrophil functions, such as exocytosis of specific granules.Selective protein phosphorylation involving protein kinase C is now generally accepted as one mechanism whereby external signals are transduced to generate specific intracellular messages that evoke distinct cell responses (1, 2). In a variety of cell types, including neutrophils (3-8), platelets (9-11), erythrocytes (12), adipocytes (13), 3T3 mouse cells (14), cells of the PC12 pheochromocytoma line (15), melanoma cells (16), and neuronal cells (17), an increase in the number of proteins phosphorylated and in the extent of 32p incorporation into both membrane and intracellular proteins appears to be related to responses to external signals. However, at the present time, neither the specific proteins phosphorylated nor their relation to a well-defined functional response has been clearly established. We have previously shown that in human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA), the phosphorylation of membrane proteins is due to the intercalation of protein kinase C into the plasma membrane (8,18) and is responsible for the release of a membrane-bound neutral proteinase and the production of superoxide anion (19). On the other hand, the degranulation responses induced by PMA or fMet-Leu-Phe are mediated by a different form of protein kinase C, the form (PKC-M) that is generated by digestion with a Ca2l-activated proteinase (calpain) (20,21) and expresses full catalytic activity in the absence of Ca2l and phospholipids (20)(21)(22)(23)(24)(25) and no longer binds to the cell membrane (21, 22).We have recently reported (26) that incubation of the cytoskeletal fraction prepared from human neutrophils with PKC-M results in the phosphorylation of a 20-kDa component of the cytoskeleton, probably representing myosin light chains. Phosphorylation of this polypeptide serves as a specific signal for its digestion by calpa...

Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease.

“…Phosphorylation of a protein or proteins at 46-48 kD has been correlated with activation ofthe respiratory burst in intact cells by several laboratories (8,(56)(57)(58). However, other data dissociates phosphorylation of a 48-kD protein(s) from stimulation of oxidative metabolism (59)(60)(61).…”

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.…”

Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease

“…1,6). Other calpain substrates affected in AD include CaM-kinase IIα (CaMK-IIα) and PKC, 2 enzymes that regulate APP phosphorylation and influence its metabolism (12)(13)(14); second messengerrelated enzymes such as phospholipase C-1, -2, -β3 (15), and cyclindependent kinase 5 (Cdk-5) (16); transcription factors such as c-Jun, c-Fos, and IκB (17,18); and the memory-related gene, cAMP regulatory element-binding protein (CREB) (19)(20)(21). Calpains also regulate cytoskeletal proteins such as spectrin (22) and MAP2 (23) and, through direct proteolytic actions and indirect modulatory effects on several protein kinases (PKC, ERK1/2, CaMK-II, and Cdk-5/p35), play a key role in regulating the dynamic behavior and turnover of cytoskeletal proteins, especially those in synapses where calpain concentrations are high (3).…”