Phosphorylation Of Plasma Membrane Proteins Research Articles

Characterization of (Ca2+ + Mg2+) adenosine triphosphatase activity and calcium transport in boar sperm plasma membrane vesicles and their relation to phosphorylation of plasma membrane proteins.

Properties of (Ca2+ + Mg2+) adenosine triphosphatase (ATPase) in plasma membranes from boar epididymal spermatozoa are described. Enzyme activity is optimum at high pH and has a high affinity for Ca2+. It is not inhibited by the calmodulin antagonist trifluoperazine (TFP), but it is inhibited by low concentrations of Ca2+. Plasma membrane vesicles obtained by hypotonic lysis of intact sperm [mixed inside-out (IOV) and right side-out (ROV) vesicles] transport 45Ca2+ in the presence of oxalate. Similar to the Ca2+-stimulated Mg ATPase activity, transport is unaffected by TFP, but unlike the ATPase, transport is at an optimum rate near neutral pH and is completely inhibited by p-chloromercurphenylsulfonate (pCMS). When plasma membranes are labeled in the presence and absence of Ca2+ and Mg2+ with [gamma-32P]ATP, differences in the intensity of labeling and lability of bound 32P to alkali and hydroxylamine suggest that two polypeptides between 100-120K may be related to a transport ATPase. The addition of TFP at concentrations which stimulate net Ca2+ uptake in intact cells causes intense labeling of a single neutrally charged protein near 68K. These labeling patterns and the properties of (Ca2+ + Mg2+) ATPase identify particular plasma membrane proteins (PMPs) from the complex surface of these cells that may be involved in Ca2+-dependent functions and support the view that calmodulin is not directly involved in the regulation of ATP-driven Ca2+ efflux from boar spermatozoa.

Somatostatin and analogs inhibit endogenous synaptic plasma membrane protein phosphorylation in vitro

The addition of somatostatin to hippocampal synaptic plasma membrane (SPM) preparations in vitro decreased subsequent phosphorylation of specific protein bands. 10 −4 M somatostatin inhibited the phosphorylation of protein bands with apparent molecular weights between 10 000 and 20 000 daltons and, to a lesser extent, 48 000 daltons (B-50) and 52 000. Increasingly greater degrees of inhibition were seen in response to somatostatin-28 and [D-Trp 8]somatostatin. Inhibition of B-50 protein phosphorylation in the presence of [D-Trp 8]somatostatin was most prominent in SPM preparations from the hippocampus and amygdala, with lesser degrees of inhibition seen in the cortex and hypothalamus. Addition of [D-Trp 8]somatostatin to an ammonium sulfate-precipitated fraction (ASP 55–80) from cortex only slightly inhibited endogenous B-50 phosphorylation. The injection of [D-Trp 8] somatostatin intracerebroventricularly into rats did not induce excessive grooming behavior but resulted in barrel rotation. These results suggest that somatostatin and congeners affect SPM protein phosphorylation in a manner different from that of ACTH, presumably involving membrane sites that bind somatostatin.

Inhibition of tyrosine protein kinases by halomethyl ketones.

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

Phosphorylated lymphocyte plasma-membrane proteins.

Lymphocytes were labelled by incubation with [32P]Pi and their plasma membranes isolated. Analysis by one-dimensional and two-dimensional gel electrophoresis revealed a small number of strongly phosphorylated polypeptides. Two of these were especially prominent; they had molecular weights of about 52000 and 90000, were acidic and were apparently not glycosylated. Similar patterns were obtained for quiescent T- and B-lymphocytes from different species and for cultured lymphoblastoid cells, although the relative amounts of the labelled polypeptides varied. Immunoprecipitation analyses of the detergent-solubilized 32P-labelled plasma membranes indicated that the glycosylated polypeptide of the human major transplantation (HLA-A and HLA-B) antigens and its mouse and pig counterparts are phosphorylated. In contrast, no phosphorylation of the membrane-associated immunoglobulin, the mouse Thy-1 antigen or the human HLA-DRw(Ia) antigen was detected. The phosphorylation patterns of human peripheral blood and nude-mouse spleen lymphocytes did not change during the period 5-30min after mitogen stimulation. Therefore a change in the phosphorylation of plasma-membrane protein(s) is probably not an early biochemical event in the initiation of T-lymphocyte and B-lymphocyte growth, although a rapid transient change cannot be ruled out. Similar plasma-membrane phosphorylation patterns were also obtained by incubating the purified plasma membrane with [gamma-32P]ATP. The phosphorylation of the 90000-mol.wt. polypeptide was particularly rapid and was stimulated by the addition of cyclic AMP.

Differences in the Cyclic AMP-Dependent Phosphorylation of Plasma Membrane Proteins of Differentiated and Undifferentiated L 6 Myogenic Cells

Differences in the cyclic AMP-dependent plasma membrane phosphorylation system of undifferentiated and differentiated L6 myogenic cells have been detected. Endogenous plasma membrane protein phosphorylation in undifferentiated L6 myoblasts was stimulated more than three fold by 5 x 10(-5) M cyclic AMP, whereas no statistically significant cyclic AMP-dependent phosphorylation of endogenous plasma membrane proteins was observed in differentiated L6 cells. In undifferentiated cells cyclic AMP promoted the phosphorylation of several proteins, the most prominent of which had a molecular weight of 110,000. In differentiated cells cyclic AMP did not selectively promote the phosphorylation of specific plasma membrane proteins. Both differentiated and undifferentiated L6 cells, however, contain a cyclic AMP-dependent protein kinase capable of catalyzing the phosphorylation of exogenous substrates, such as histone f2b. Therefore, the data show that differentiation in L6 cells is associated with a selective change in the activity of a plasma membrane cyclic AMP-dependent protein kinase which employs endogenous membrane proteins as substrate.

Cyclic Adenosine 3′,5′-Monophosphate-Independent Phosphorylation of Externally Oriented Plasma Membrane Proteins in Transformed and Nontransformed Mouse Embryo Cell Lines2

Thirty transformed and nontransformed mouse embryo cell lines were analyzed to determine if increased plasma membrane phosphorylation is a characteristic of all transformed cell lines. Nontransformed, virally and chemically transformed, and smooth surface-transformed cell lines derived from Swiss, BALB/c, AKR, or C3H mice were studied. Cyclic AMP-independent phosphorylation of plasma membrane proteins was comparable in nontransformed and simian virus 40 (SV40)-transformed Balb/3T3 cells, in nontransformed and 3-methylcholanthrene (MCA)-transformed C3H/10T 1/2 cells, and in nontransformed and MCA-transformed AKR-2B cells. In contrast, increased plasma membrane phosphorylation was observed in MCA- and smoooth surface-transformed Balb/3T3 cells and in SV40-transformed Swiss 3T3 cells compared to the respective nontransformed parent cell lines. These observations suggest that increased cyclic AMP-independent phosphorylation of externally oriented plasma membrane proteins is not a universal marker for the transformed phenotype.

Pituitary hormones influence polyphosphoinositide metabolism in rat brain

In a variety of tissues an enhanced metabolism of phosphatidyl inositol (PI) is observed after receptor activation by hormones or neurotransmitters which utilize calcium as second messenger [ 1,2]. The enhancement is manifested by an increased rate of incorporation of Pi into PI and is frequently accompanied by an increase in labelling of phosphatidic acid (PA) [ 11. It is thought to reflect an increased breakdown of PI to diacylglycerol within the plasma membrane, which is then rapidly phosphorylated to PA and consequently transformed into PI in the endoplasmic reticulum. With respect to nervous tissue, the rapid metabolism of the polyphosphoinositides (DPI, phosphatidyl-myoinositol4-phosphate; TPI, phosphatidylmyoinositol4,5-diphosphate) especially suggests an important effect in membrane function. In contrast to the low quantities of DPI and TPI in membranes from a variety of cell types, brain cell plasma membranes contain appreciable amounts of these polyphosphoinositides [3]. It appears that they play a role in the Ca2+ influx into the cell [ 1,2,4]. In peripheral tissues and cells it was demonstrated that peptides (vasopressin, insulin, substance P) may regulate the metabolism of cell membrane phosphatidyl inositides [ 5-81. In view of these findings and our recent results on the regulatory role of peptide hormones and fragments of peptide hormones on synaptic plasma membrane protein phosphorylation [9,10], it was of interest to investigate whether such peptides influence brain membrane phospholipid metabolism.

A GTP-specific protein kinase in plasma membranes of mouse fibroblasts

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence of [γ- 32P]GTP. The phosphoprotein pattern showed one main component with 100 k which could not be phosphorylated with ATP. Optimal phosphorylation of pp 100 was dependent on protein concentration. Strict substrate specificity of the corresponding endogenous kinase could be demonstrated by comparative electrophoresis with a plasma membrane sample phosphorylated with [γ- 32P]ATP and by isotope dilution experiments. GTP-specific phosphorylation of pp 100 was not dependent on cyclic nucleotides but influenced by GDP, GMP and AMP.

Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins: Antagonistic eeffects of cyclic AMP and cyclic GMP

32P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E 1 one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP. Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes. These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.

Short-time phosphorylation of plasma membrane proteins by endogenous kinases

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence and absence of cyclic nucleotides. Using low concentrations of membrane protein the kinetics of ATP-dependent 32P-incorporation showed a rapid phosphorylation reaction up to 2 sec of incubation which was stimulated by cAMP and inhibited by cGMP. This short-time phosphorylation reaction was followed by a rapid dephosphorylation and a slower rephosphorylation. This phenomenon was dependent on protein concentration.

Regulation of plasma membrane protein phosphorylation in two mammalian cell types

The appreciation of protein phosphorylation as a ubiquitous mechanism for the post-translational control of protein function has drawn our attention to the phosphorylation of plasma membrane proteins. We have studied this phenomenon in the human erythrocyte and rat adipocyte, and have observed several features, common to the two systems, which may be of general significance. In examining protein phosphorylation in intact cells incubated with 32Pi, it is evident that the 32P-polypeptides of the plasma membrane are among the most highly labelled species in the cell, despite their minor contribution to overall protein content. The addition of epinephrine (to adipocytes) or cAMP (to erythrocytes) increases the phosphorylation of certain peptides, whereas others are unaffected. The protein kinases mediating these phosphorylations are present in the plasma membrane as isolated, and can be divided into two groups--cAMP dependent and cAMP independent. These two classes of kinase differ markedly in their substrate specificity toward endogenous and exogenous polypeptide substrates. Two classes of protein kinases with similar properties can be detected in the cytoplasm. The relationship between the membrane-bound and cytoplasmic enzymes is uncertain. The potential roles of the plasma membrane cAMP dependent protein kinases are evident from the diverse effects of cAMP on surface properties; however, the prevalence of plasma membrane proteins phosphorylated via cAMP independent pathways is striking. Thus, elucidation of the regulatory properties of the plasma membrane cAMP independent protein kinases may give new insight into the control of a variety of surface phenomena not mediated by cAMP.

Cyclic AMP-dependent reversible phosphorylation of renal medullary plasma membrane protein.

In medullary tissue from bovine kidney, three elements required for cyclic AMPdependent phosphorylation of plasma membrane protein were found: a) a protein kinase which is stimulated by cyclic AMP (Km = 10-7M) ; b) a plasma membrane fraction containing protein(s) which could serve as an acceptor of phosphate and which could be phosphorylated through the action of cyclic AMP—dependent protein kinase; and c) a protein phosphatase, which catalyzes the release of phosphate bound to membrane proteins and which is not stimulated by cyclic AMP. These findings support the view, that cyclic AMP—dependent phosphorylation of plasma membrane protein(s) might be the step in the cellular mechanism of ADH action subsequent to the ADH—stimulated formation of cyclic AMP.(Endocrinology 91: 738, 1972)