Phosphorylation Of Endogenous Membrane Proteins Research Articles

Enhanced membrane protein kinase C activity in myocardial ischemia.

The protein kinase activity in cytosol was similar in control, ischemic, and reperfused hearts; however, a 1.5-fold increase in membrane protein kinase activity was induced by ischemia and reperfusion. The H-7 inhibitable cytosolic protein kinase activity decreased by 40% with 30 min ischemia, while that of membrane fraction increased 1.8-fold. However, the CGS9343B inhibitable protein kinase activity in cytosolic fractions was unaffected by ischemia, while that of membrane increased by about 1.7-fold. These results suggest that myocardial ischemia is associated with enhanced protein kinase C and calmodulin-dependent kinase activities in membrane fraction. Furthermore, the results also suggest a translocation of protein kinase C activity from the cytosol to the membrane. Reperfusion of ischemic myocardium did not result in any further increase of protein kinase C and calmodulin-dependent kinase activities in the membrane. These enhanced protein kinase activities also resulted in an enhanced phosphorylation of endogenous membrane proteins. The creatine kinase released from the heart was increased by both ischemia and reperfusion. Therefore, these results suggest that biochemical cascades of reactions caused by enhanced membrane protein kinase C and calmodulin-dependent kinase activities may contribute to ischemic-reperfusion injury.

Membrane-associated protein kinase activities in seminal plasma from vasectomized men.

Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.

Effect of protein kinase P on phosphorylations catalyzed by the epidermal growth factor.

Protein kinase P (PK-P) activated by histones or certain other basic compounds has been purified previously from yeast [Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N. & Racker, E. (1987) Proc. Natl. Acad. Sci. USA 84, 925-929]. It is shown here that PK-P is present in solubilized membranes of A-431 carcinoma cells where it changes the epidermal growth factor (EGF) receptor kinase activity. Polylysine, a weak PK-P activator, inhibited the autophosphorylation of the EGF receptor both in the absence and presence of EGF. Increased PK-P activity induced by histone 1, a potent activator, gave rise to increased autophosphorylation of the EGF receptor as well as phosphorylation at tyrosine residues of numerous other endogenous membrane components. The stimulation by histone was particularly striking in the presence of EGF. A similar stimulation was achieved with polylysine and EGF on addition of yeast PK-P. However, addition of yeast PK-P in the presence of histone 1 markedly inhibited the EGF-stimulated phosphorylation of endogenous membrane proteins. We conclude from these results that the effect of PK-P on the EGF receptor takes place in three phases: at low levels PK-P inhibits the autophosphorylation, at intermediate levels it stimulates the autophosphorylation as well as the EGF-dependent phosphorylation of numerous other membrane proteins, and at high levels it inhibits the phosphorylation of these proteins.

Enhanced phosphorylation of endogenous membrane proteins during induction of B lymphocyte proliferation

Phosphorylation of endogenous proteins was assessed employing membrane preparations derived from splenocytes induced to proliferate in response to Sepharose linked anti-immunoglobulins. Time course studies revealed that enhanced protein phosphorylation was preceded by cell enlargement and was either followed by or closely related in time to the onset of DNA synthesis. Thus maximal enhancement of phosphorylation was initially observed at 24 h whereas cell enlargement was optimal at 16 h at a time when there was no enhancement in protein phosphorylation. Furthermore thymidine incorporation was maximal at 32 h and low at 24 h when phosphoprotein synthesis was maximally enhanced. Taken together, these results suggest that phosphorylation of endogenous membrane proteins may be involved in signalling entry of cells into S phase of the cell cycle.

Characterization of the interaction of 5'-p-fluorosulfonylbenzoyl adenosine with the epidermal growth factor receptor/protein kinase in A431 cell membranes.

Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.

Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor.

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.

A non-mitogenic analogue of epidermal growth factor enhances the phosphorylation of endogenous membrane proteins

The cyanogen bromide cleaved analogue of Epidermal Growth Factor (EGF) binds to EGF receptors with reduced affinity but fails to induce DNA synthesis. However, at similar receptor occupancy, cyanogen bromide cleaved EGF is as potent as EGF in enhancing the phosphorylation of endogenous membrane proteins mediated by the EGF specific, cyclic nucleotide independent protein kinase. Two possibilities arise concerning the biological role of the EGF induced membrane protein phosphorylation: 1) it is not related to the triggering of DNA synthesis by EGF and does not serve as “second messenger” for the growth factor. 2) it is a necessary but not sufficient biochemical signal for the activation of DNA synthesis by EGF.

Endogenous protein phosphorylation in isolated plasma membranes of AH-66 hepatoma ascites cells.

The phosphorylation of endogenous membrane proteins by an endogenous protein kinase was studied in isolated plasma membranes from AH-66 hepatoma ascites cells using [gamma-32P]ATP as a precursor. The phosphorylation occurred very rapidly in the presence of 10 mM Mg2+ and reached a maximal level at 2 min. Ca2+ strongly inhibited the phosphorylation reaction and antagonized the activation produced by Mg2+. Neither cyclic AMP nor cyclic GMP had a significant effect on the phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that only membrane proteins ranging in molecular weight from 125,000 to 200,000 were heavily phosphorylated and those of less than 60,000 molecular weight were not phosphorylated at all. The protein kinase activity was readily extractable from the plasma membranes with 1 mM EDTA at pH 8.5. Among exogenous substrates, the extracted protein kinase catalyzed the phosphorylation of histone, protamine and phosvitin rather than casein. When the extracted protein kinase was subjected to chromatography on DEAE-Sepharose, a single major peak of cyclic AMP-independent protein kinase was eluted at a position quite different from those of the cytosolic protein kinases.

Behavioral and regional neurochemical sequelae of hippocampal destruction in the rat

In an attempt to determine the nature of changes found in selected remaining brain regions following hippocampal damage, endogenous in vitro protein phosphorylation was measured in membranes obtained from n. accumbens, n. caudatus, septal area, hypothalamus and frontal neocortex in rats with bilateral hippocampal destruction, animals with damage limited to postero-lateral neocortex, and in sham operated subjects. Measurements were made in separate groups of animals 8 and 28 days after surgery. The membrane protein profiles measured by SDS polyacrylamide slab gel electrophoresis from tissues obtained from all groups were remarkably similar as were the phosphoprotein profiles. The most prominent bands for phosphoprotein activity had estimated molecular weights of 43K, 48K, 50K, and 82K dalton. Specific changes were found after the two forms of brain damage and at the two postoperative times at which the animals were sacrificed. Eight days after surgery an enhanced endogenous phosphorylation was found in the caudate and accumbens samples in the hippocampectomized group, whereas no differences were seen in these areas between cortically lesioned and sham operated rats. Twenty eight days after surgery, both brain lesioned groups displayed an enhanced endogenous phosphorylation of membrane proteins compared to sham operates in these regions. In addition, changes were also found in the neocortical samples. These results indicate that there are neurochemical changes occurring over time after surgery. The changes in membrane phosphoproteins may be related to altered synaptic efficacy. Novelty-induced and water immersion-induced excessive grooming were also studied in these animals. Animals with hippocampal destruction tended to groom less than other animals at both postoperative periods but were not different from other animals when tested for excessive grooming after water immersion.

Changes in synaptic membrane phosphorylation after tetanic stimulation in the dentate area of the rat hippocampal slice

slices of rat brain hippocampus were stimulated electrically in the perforant pathway. After electrical stimulation, known to produce long-lasting post-tetanic potentiation, endogenous phosphorylation of membrane proteins was measured in a crude mitochondrial fraction, prepared from stimulated and unstimulated slices. Tetanic stimulation specifically enhanced the incorporation of [ 32P]phosphate into a protein band with apparent molecular weight of 50K. When the same number of pulses were given, but at a much slower rate (1 pulse per 4 sec instead of 15 pulses per sec) no post-tetanic stimulation and concomitantly, no enhanced incorporation of [ 32P]phosphate were observed into the 50K band. When the stimulation of the slices was performed in Ca 2+-free medium, again no potentiation and no enhanced incorporation into the 50K protein band were observed. It is suggested that electrical stimulation enhances the activity of the protein kinase that phosphorylates the 50K protein.

Endogenous phosphorylation of membrane proteins in normal and in hereditary spherocytosis erythrocytes

The phosphorylation state of the proteins in hereditary spherocytosis erythrocyte membranes, incubated in the presence of [ γ- 32P]ATP, appears to be different from that in normal ones. This is indicated by the finding that in the two types of erythrocyte membranes the ratios between the 32P-labeling of their phosphorylserine and phosphorylthreonine residues were different.

Association of decreased membrane protein phosphorylation with red blood cell spherocytosis

A close association between sphering of human red cells and deficient phosphorylation of their membrane proteins has been documented in three spearate situations. Red cells rendered spheroidal by exposure to: (1) elevated temperatures; (2) two sulfhydryl inhibitors (N-ethylmaleimide or paramercuribenzoate); or (3) in the genetic situation of hereditary spherocytosis--all manifest deficient phosphorylation of endogenous membrane proteins by ATP. In the two former cases, we have noted an exact association between the onset of red cell sphering (e.g., as temperatures rose above 48 degrees C or N-ethylmaleimide concentrations exceeded 2 mumoles/ml RBC) and the development of deficient ghost protein phosphorylation.

Association of Decreased Membrane Protein Phosphorylation With Red Blood Cell Spherocytosis

A close association between sphering of human red cells and deficient phosphorylation of their membrane proteins has been documented in three spearate situations. Red cells rendered spheroidal by exposure to: (1) elevated temperatures; (2) two sulfhydryl inhibitors (N-ethylmaleimide or paramercuribenzoate); or (3) in the genetic situation of hereditary spherocytosis--all manifest deficient phosphorylation of endogenous membrane proteins by ATP. In the two former cases, we have noted an exact association between the onset of red cell sphering (e.g., as temperatures rose above 48 degrees C or N-ethylmaleimide concentrations exceeded 2 mumoles/ml RBC) and the development of deficient ghost protein phosphorylation.

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 Mg 2+-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.

Phosphorylation of Endogenous Membrane Proteins by Endogenous Protein Kinase at the Outer Surface of Ehrlich Cells

An endogenous protein kinase at the surface of Ehrlich cells has been studied. Using exogenous (gamma32P)ATP as a phosphoryl group donator, a transfer was demonstrated into endogenous acceptor protein(s) as well as to exogenous phosvitin. Seryl- and threonyl-residues isolated from the endogenous and exogenous acceptor protein were found to be labeled. The ratio between the labeled phosphorylserine and phosphorylthreonine was about 3.5:1 for both the endogenous acceptor of the intact cells and the exogenous acceptor. In similar experiments with a membrane preparation from Ehrlich cells, this ratio increased to about 7:1 provided the exogenous acceptor protein was absent. The results were independent of whether 1 X 10(-5) M dibutyryl cyclic AMP was used or not with intact cells and a membrane fraction mainly consisting of vesicles. Whether the regulatory subunit of the membrane-associated protein kinase was in cis- or trans-disposition to the catalytic subunit no binding and dependence of the cyclic nucleotide was observed. Since the purified membrane fraction was considered free from endogenous cyclic AMP, it was concluded that the membrane-associated protein kinase of Ehrlich cells is not dependent on cyclic AMP. The critical role of arginine for the cyclic AMP dependence of the serine-containing residue in the catalytic subunit is discussed.

Muscle membrane protein kinase in myotonic muscular dystrophy.

MYOTONIC muscular dystrophy is an inherited disorder of man with manifestations in many organ systems including skeletal muscle myotonia and dystrophy, testicular atrophy, cataract formation, hypercatabolism of IgG, cranial bone malformations, and glucose intolerance with enhanced insulin levels (see ref. 1 for clinical references). Our previous experiments demonstrated a significant diminution in the phosphorylation of endogenous membrane proteins of frozen erythrocyte ghosts from myotonic patients1. In subsequent studies the technique of electron magnetic resonance was employed to compare the erythrocyte membrane milieu of normal and myotonic paients2. Following incorporation of 5-nitroxide methyl stearate into erythrocyte membranes, the spin label was located in a less polar and somewhat more fluid region in myotonic membranes than in normal membranes. Although such experiments do not specify the molecular membrane abnormality or its possible relationship to protein phosphorylation, they do support the suggestion that myotonic muscular dystrophy is a disease resulting from a basic membrane abnormality.