A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ

A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.     

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates

Summary The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examining competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s).The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP.Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.     

Phosphorylation of rabbit and human erythrocyte membranes by soluble adenosine 3':5'-monophosphate-dependent and -independent protein kinases.

Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes

Incorporation of 32P from [gamma-32P]ATP into tyrosine residues of the insulin-like growth factor (IGF)-II receptor was observed in a Triton X-100-insoluble fraction of rat adipocyte plasma membranes. IGF-II receptor phosphorylation proceeded to a stoichiometry of approximately 0.5 mol of phosphate/IGF-II binding site after 10 min of incubation at 4 degrees C. A Km for ATP of 6 microM was calculated for this phosphorylation reaction. Addition of IGF-II caused an approximately 2-fold increase in tyrosine phosphorylation of the IGF-II receptor in this preparation. In contrast, phosphorylation of angiotensin II by the Triton X-100 washed membranes was not stimulated by IGF-II. Incubation of purified receptor immobilized on IGF-II agarose or of receptor-enriched low density microsomal membranes with [gamma-32P]ATP did not result in appreciable incorporation of [32P]phosphate into the IGF-II receptor nor into exogenous substrates. These data suggest that the IGF-II receptor is not a tyrosine protein kinase capable of autophosphorylation but that it is a substrate for a tyrosine protein kinase endogenous to the adipocyte plasma membrane. The stimulatory effect of IGF-II on the tyrosine phosphorylation of its receptor may be due to a conformational change which converts the receptor to a better substrate for this tyrosine kinase.     

Extraction of peripheral proteins from nicotinic acetylcholine receptor-enriched membranes

The solubilisation of membrane proteins from nicotinic acetylcholine receptor-enriched membranes from the electric organ of Torpedo marmorata was studied. Chaotropic ions were shown to be ineffective in extracting peripheral proteins from these membranes. Two different anhydrides, 2,3-dimethylmaleic and 3,4,5,6-tetrahydrophthalic anhydride, released certain peripheral membrane proteins but not the integral receptor protein. Treatment of membranes containing $\text{> 3}\text{nmol}$ α-bungarotoxin binding sites per mg protein with anhydride resulted in a 43 kDa polypeptide as the major constituent of the solubilised material. The nature of the 43 kDa polypeptide is discussed. Gentle anhydride treatment did not change the α-bungarotoxin and carbamoylcholine binding properties of the receptor.     

Phosphorylation of isolated plasma membranes of AH-66 hepatoma ascites cells by casein kinase 1

The isolated plasma membranes of AH-66 hepatoma cells were phosphorylated by casein kinase 1 purified from the cytosol fraction of AH-66 cells. Casein kinase 2 purified from the same source had little effect on the phosphorylation of the plasma membranes. Two-dimensional gel electrophoresis and autoradiography showed that casein kinase 1 enhanced the phosphorylation of approx. 10 plasma membrane proteins that are phosphorylated only faintly in the isolated plasma membranes by endogenous protein kinase. Among these phosphoproteins, tubulin was identified as judged from their molecular weights and isoelectric points. These results suggest that one of the physiological functions of casein kinase 1 is phosphorylation of plasma membrane and plasma membrane-associated proteins.     

Calcium- and Calmodulin-Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor-Rich Membranes from Electroplax of Narke japonica

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.

When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Protein phosphatase activity in acetylcholine receptor-enriched membranes.

Phosphorylation of the acetylcholine receptor (AChR)¹ has been demonstrated in AChR-enriched membranes prepared from the electric organ of $\text{Torpedo californica}$. In this report we show that the same AChR-enriched membrane fraction also contains phosphoprotein phosphatase activity which dephosphorylates both the endogenous AChR and exogenous phosphorylated casein. Release of [³²P] PO₄ from phosphorylated casein was shown to be inhibited by F^? as well as GTP. cAMP and cGMP were without effect. Dephosphorylation of the membrane-bound AChR was also inhibited by F^?. This phosphorylation-dephosphorylation mechanism may play a role in mediating the function of the AChR at the synapse.     

Phosphorylation of the 165-kDa dihydropyridine/phenylalkylamine receptor from skeletal muscle by protein kinase C

Dihydropyridine-sensitive Ca2+ channels exist in many different types of cells and are believed to be regulated by various protein phosphorylation and dephosphorylation reactions. The present study concerns the phosphorylation of a putative component of dihydropyridine-sensitive Ca2+ channels by the calcium and phospholipid-dependent protein kinase, protein kinase C. A skeletal muscle peptide of 165 kDa, which is known to contain receptors for dihydropyridines, phenylalkylamines, and other Ca2+ channel effectors, was found to be an efficient substrate for protein kinase C when the peptide was phosphorylated in its membrane-bound state. Protein kinase C incorporated 1.5-2.0 mol of phosphate/mol of peptide within 2 min into the 165-kDa peptide in incubations carried out at 37 degrees C. In contrast to the membrane-bound peptide, the purified 165-kDa peptide in detergent solution was phosphorylated to a markedly less extent than its membrane-bound counterpart; less than 0.1 mol of phosphate/mol of peptide was incorporated. Preincubation of the membranes with several types of drugs known to be Ca2+ channel activators or inhibitors had no specific effects on the rate and/or extent of phosphorylation of the 165-kDa peptide by protein kinase C. The phosphorylation of the membrane-bound 165-kDa peptide by protein kinase C was compared to that catalyzed by cAMP-dependent protein kinase and was found to be not additive. Prior phosphorylation of the 165-kDa peptide by cAMP-dependent protein kinase prevented subsequent phosphorylation of the peptide by protein kinase C. Phosphoamino acid analysis indicated that protein kinase C phosphorylated the 165-kDa peptide at both serine and threonine residues. Phosphopeptide mapping experiments showed that protein kinase C phosphorylated one unique site in the 165-kDa peptide, and, in addition, other sites that were phosphorylated by either cAMP-dependent protein kinase or a multifunctional Ca2+/calmodulin-dependent protein kinase. The results suggest that the 165-kDa dihydropyridine/phenylalkylamine receptor could serve as a physiological substrate of protein kinase C in intact cells. It is therefore possible that the regulation of dihydropyridine-sensitive Ca2+ channels by activators of protein kinase C may occur at the level of this peptide.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.     

Protein kinase associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates

When highly purified myelin from rat sciatic nerve was incubated with [γ-³²P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and ³²P from [γ-³²P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg²⁺-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3′,5′-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing.From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Identification of the phosphorylation sequence in the cytoplasmic tail of the varicella-zoster virus Fc receptor glycoprotein gpI.

Varicella-zoster virus (VZV) glycoprotein gpI, the homolog of herpes simplex virus gE, functions as a receptor for the Fc portion of immunoglobulin G. Like other cell surface receptors, this viral receptor is highly phosphorylated in cell culture. To identify the precise location of the cellular kinase-mediated phosphorylation, we generated a tailless deletion mutant and several point mutants which had altered serine and threonine residues within the cytoplasmic domain of gpI. The mutated and wild-type genes of gpI were transfected and expressed within a vaccinia virus-T7 polymerase transfection system in order to determine what effect these mutations had on the phosphorylation state of the protein in vivo and in vitro. Truncation of the cytoplasmic domain of gpI diminished the phosphorylation of gpI in vivo. Examination of the point mutants established that the major phosphorylation sequence of gpI was located between amino acids 593 and 598, a site which included four phosphorylatable serine and threonine residues. Phosphorylation analyses of the mutant and wild-type glycoproteins confirmed that gpI was a substrate for casein kinase II, with threonines 596 and 598 being critical residues. Although the mutant glycoproteins were phosphorylated by casein kinase I, protease V8 partial digestion profiles suggested that casein kinase II exerted the major effect. Thus, these mutagenesis studies demonstrated that the gpI cytoplasmic sequence Ser-Glu-Ser-Thr-Asp-Thr was phosphorylated in mammalian cells in the absence of any other herpesvirus products. Since the region defined by transfection was consistent with results obtained with in vitro phosphorylation by casein kinase II, we propose that VZV gpI is a physiologic substrate for casein kinase II. Immunofluorescence and pulse-chase experiments demonstrated that the mutant glycoproteins were processed and transported to the outer cell membrane.     

Phosphorylation and dephosphorylation of spectrin

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of ³²Pi from [³²P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [³²P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.     

Major vault protein is a substrate of endogenous protein kinases in CHO and PC12 cells.

Major vault protein (MVP) is the predominant member of a large cytosolic ribonucleoprotein particle, termed vault. We have previously shown that MVP derived from electric ray electric organ becomes phosphorylated by protein kinase C in vitro and by tyrosine kinase in vivo. Here we show that MVP from two mammalian cell lines (CHO and PC12 cell) becomes highly phosphorylated by endogenous protein kinases in cell-free systems. The susceptibility to protein kinases differs substantially from those observed in MVP derived from electric organ. Phosphorylation of MVP depends on the presence of Mg2+ and can be inhibited by the chelating agent EDTA. Inhibitors of casein kinase II attenuate the phosphorylation of MVP. In contrast to CHO cells, addition of recombinant casein kinase II enhances the phosphorylation of MVP in PC12 cells. Endogenous kinase activity is of particulate nature and copurifies with vault particles. Immuno-affinity purified vaults containing recombinant tagged MVP expressed in CHO cells reveal no autophosphorylation, suggesting that protein kinase activity is not an intrinsic property of vaults. Our results suggest that cell-specific phosphorylation of MVP may play a critical role in vault function.     

Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other hand, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)