Carbachol-induced protein phosphorylation changes in bovine tracheal smooth muscle

The changes in protein phosphorylation associated with bovine tracheal smooth muscle contraction were studied by labeling intact muscle strips with [32P]PO4(3-) and analyzing the phosphoproteins by two-dimensional gel electrophoresis. Among 20 to 30 phosphoproteins resolvable with the two-dimensional electrophoresis system, the phosphorylation of 12 proteins was reproducibly affected by treatment with carbachol, in a time-dependent manner. Five of these proteins have been identified as 20-kDa myosin light chain, caldesmon, synemin, and two isoelectric variants of desmin. The other 7 are low molecular weight (Mr less than 40,000) cytosolic proteins. One cytosolic protein and myosin light chain are quickly but transiently phosphorylated by carbachol, the peak of myosin light chain phosphorylation being at about 1 min after agonist addition. In contrast, both variants of desmin, synemin, caldesmon, and 5 cytosolic proteins are phosphorylated at varying rates and remain phosphorylated for the duration of carbachol action. These "late" phosphorylation changes occur simultaneously with the dephosphorylation of one cytosolic protein. These carbachol-induced phosphorylation changes, like the contractile response, appear to be calcium-dependent. The addition of 12-deoxyphorbol 13-isobutyrate, a protein kinase C activator, causes a dose-dependent, sustained contraction of tracheal smooth muscle which develops more slowly than that induced by carbachol. This contractile response is associated with the same protein phosphorylation changes as those observed after prolonged carbachol treatment. In contrast, forskolin, an adenylate cyclase activator and a potent smooth muscle relaxant, induces the phosphorylation protein 3 and one variant of desmin. These observations strongly suggest that different phosphoproteins may be mediators of tension development and tension maintenance in agonist-induced contraction of tracheal smooth muscle.     

Ca2+-dependent phosphorylation of rat ovary proteins

A 105,000 × g supernatant fraction from prepubertal rat ovaries was incubated in the presence of [γ-³²P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Inclusion of Ca²⁺ in the phosphorylation reaction promoted a selective ³²p incorporation into two proteins of M_r = 95,000 and 50,000. Inclusion of chlorpromazine with Ca²⁺ blocked the Ca²⁺-stimulated increase of ³²p incorporation. Our results demonstrate the presence of Ca²⁺-stimulated protein phosphorylation system capable of recognizing endogenous substrate proteins in the prepubertal rat ovary.     

Protein kinase activity in cell free extracts of Streptomyces avermitilis; comparing wild type and overproducing mutant

Summary Protein kinase activity was assayed in cell-free extracts prepared from mycelia of a wild strain (MA) and an overproducing mutant (RX 2) of Streptomyces avermitilis. At least 10 different polypeptides ranging in M _r from 10 000 to 120 000 were found to be phosphorylated on analysis of ³²P labeled cell lysates by gel electrophoresis and phosphorimage. The protein profile and the level of phosphorylation varied depending on the culture stage of the mycelia.     

Lack of phosphate incorporation into TN-I in live frog muscle

The phosphorylation of TN-I was investigated in muscles of live frogs injected with [³²P]orthophosphate. Isolation of TN-I was carried out by the rapid and specific technique of affinity chromatography developed by Syska, Perry, and Trayer [FEBS Lett.40, 253–257 (1974)] followed by gel electrophoresis in the presence of sodium dodecyl sulfate. No significant labeling of TN-I was found even in frogs which were exposed to the ³²P-treatment for several days. A comparison of the specific radioactivity of TN-I from resting and contracting frog muscle showed no change in the ³²P content of TN-I during muscle contraction.     

Contraction in intact pig aortic strips is not always associated with phosphorylation of myosin light chains.

Intact pig aortic strips were incubated in medium containing [32P]P1 and various Ca2+ concentrations. The 32P content of the myosin P-light chain was determined by radioautography after electrophoresis in the presence of sodium dodecyl sulphate. Although treatment of the strips with noradrenaline always caused a rise in tension, this was not necessarily accompanied by increased phosphorylation of the P-light chain. These results indicate that, in aortic smooth muscle, phosphorylation of the P-light chain is not obligatory for contraction.     

Evidence for phosphorylation of rat brain guanylate cyclase by cyclic AMP-dependent protein kinase

Direct phosphorylation of purified rat brain guanylate cyclase by cyclic AMP-dependent protein kinase is demonstrated. In the presence of [γ-³²P]ATP, ³²P was incorporated into the protein to the extent of 0.8 to 0.9 mol/mol of guanylate cyclase. The presence of ³²P in the guanylate cyclase molecule was demonstrated by gel-filtration and by autoradiography after gel electrophoresis. The phosphorylation was accompanied by an increase in enzyme activity, characterized by an increase of V_M. These results suggest that the activity of guanylate cyclase may be regulated in vivo by phosphorylation.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Endogenous phosphorylation of platelet membrane proteins.

The characteristics of the phosphorylating activity of platelet membranes have been studied. Plasma membranes of human platelets isolated by the glycerol lysis technique were shown to incorporate significant amounts of [³²P]phosphate into specific membrane proteins. This activity was only partially cyclic 3′:5′-monophosphate (cyclic AMP)-dependent but had most of the other characteristics of protein kinases derived from other sources. Maximal stimulation of endogenous phosphorylation was obtained at 1 × 10^?7, m cyclic AMP and exceeded by approximately 30% the [³²P]phosphate incorporation in the absence of this cyclic nucleotide. The platelet membrane protein kinase was able to phosphorylate exogenous proteins, e.g., histone, fibrinogen etc., as well as endogenous membrane proteins. The latter solubilized by sodium dodecyl sulfate and separated by dodecyl sulfate-polyacrylamide gel electrophoresis incorporated [³²P]phosphate into three polypeptides of apparent molecular weights 52,000, 31,000, and 20,000. The phosphorylation of the polypeptide of molecular weight 52,000 was cyclic AMP-dependent.     

Vinculin Phosphorylation at Tyr1065 Regulates Vinculin Conformation and Tension Development in Airway Smooth Muscle Tissues

Vinculin localizes to membrane adhesion junctions in smooth muscle tissues, where its head domain binds to talin and its tail domain binds to filamentous actin, thus linking actin filaments to the extracellular matrix. Vinculin can assume a closed conformation, in which the head and tail domains bind to each other and mask the binding sites for actin and talin, and an open activated conformation that exposes the binding sites for talin and actin. Acetylcholine stimulation of tracheal smooth muscle tissues induces the recruitment of vinculin to the cell membrane and its interaction with talin and actin, which is required for active tension development. Vinculin phosphorylation at Tyr¹⁰⁶⁵ on its C terminus increases concurrently with tension development in tracheal smooth muscle tissues. In the present study, the role of vinculin phosphorylation at Tyr¹⁰⁶⁵ in regulating the conformation and function of vinculin during airway smooth muscle contraction was evaluated. Vinculin constructs with point mutations at Tyr¹⁰⁶⁵ (vinculin Y1065F and vinculin Y1065E) and vinculin conformation-sensitive FRET probes were expressed in smooth muscle tissues to determine how Tyr¹⁰⁶⁵ phosphorylation affects smooth muscle contraction and the conformation and cellular functions of vinculin. The results show that vinculin phosphorylation at tyrosine 1065 is required for normal tension generation in airway smooth muscle during contractile stimulation and that Tyr¹⁰⁶⁵ phosphorylation regulates the conformation and scaffolding activity of the vinculin molecule. We conclude that the phosphorylation of vinculin at tyrosine 1065 provides a mechanism for regulating the function of vinculin in airway smooth muscle in response to contractile stimulation.     

Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues

The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [³²P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of ³²P incorporation and the electrophoretic patterns were dependent on ³²P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K_m values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.     

Evidence for Phosphorylation of the Extracellular Acid Phosphatase of Leishmania donovani1

The presence of two phosphorylated molecular species in the culture supernatants of axenically cultivated Leishmania donovani promastigotes was demonstrated by biosynthetically labeling cultures with [³²P]phosphate. One of these species was resolved into two bands with M_r's of 149,000 and 97,000 by dissociating polyacrylamide gel electrophoresis and copurified with the extracellular acid phosphatase activity produced by the promastigotes. The site of phosphorylation of the extracellular acid phosphatase is not yet known.     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

Factor-free and one-factor-promoted poly(U,C)-dependent synthesis of polypeptides in cell-free systems from Escherichia coli

α-Tropomyosin from rat cardiac muscle was shown by two-dimensional gel electrophoresis to become phosphorylated when tissue slices were incubated in Eagle's medium supplemented with ³²P_i. In the adult rat and mouse heart the level of phosphorylation was ～30%, but the level was much higher in the foetal heart (60–70%). A similar developmental trend was observed in skeletal muscle from the rat and mouse, where phosphorylated forms of both α- and β-tropomyosins were observed. When rat cardiac cells were grown in tissue culture in the presence of ³²P_i, radioactivity was incorporated into the region of the gel containing tropomyosin.     

Cyclosporine-induced renal artery smooth muscle contraction is associated with increases in the phosphorylation of specific contractile regulatory proteins

Cyclosporine A (CSA) is a type 2B phosphatase inhibitor which can induce contraction of renal artery smooth muscle. In this investigation, we examined the phosphorylation events associated with CSA-induced contraction of bovine renal artery smooth muscle. Contractile responses were determined in a muscle bath and the corresponding phosphorylation events were determined with whole cell phosphorylation and two-dimensional gel electrophoresis. CSA-induced contractions were associated with increases in the phosphorylation of the 20 kDa myosin light chains (MLC20) and different isoforms of the small heat shock protein, HSP27. Cyclic nucleotide-dependent relaxation of CSA-induced contractions was associated with increases in the phosphorylation of another small heat shock protein, HSP20, and decreases in the phosphorylation of the MLC20, and some isoforms of HSP27. These data suggest that CSA-induced contraction and relaxation of vascular smooth muscle is associated with increases in the phosphorylation of specific contractile regulatory proteins.     

Human osteogenic sarcoma cells exhibit enhanced protein phosphorylation

Protein phosphorylation was compared in normal human cells and human osteogenic sarcoma cells. The phosphorylation of endogenous cellular protein substrates was measured by two independent methods, incubation of homogenized cells with [γ-³²P]ATP or labeling of intact cells with Na₂H³²PO₄. Phosphorylated proteins were identified by SDS-polyacrylamide gel electrophoresis and autoradiography. The stained protein bands of all four osteosarcoma cell lines were nearly identical to those of the normal cells. However, each of the osteosarcoma cell lines showed autoradiographic evidence of enhanced phosphorylation in many different protein bands which was neither cyclic AMP-dependent nor a function of cellular growth rate or density. When normal and tumor cell homogenates were mixed prior to incubation with [γ-³²P]ATP, the resulting phosphoprotein patterns resembled those obtained with the tumor cells alone. In addition, a surgically derived osteogenic sarcoma was cultured and an established line obtained; another portion of the fresh tumor was immediately homogenized and used in a phosphorylation assay. The same enhanced phosphorylation pattern was obtained with the homogenized fresh tumor as with the cell line established from it. These results suggest thathuman osteogenic sarcoma cells are able to perform a significantly increased amount of phosphorylation of endegenous cellular protein substrates when compared to normal human cells.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Guard of Delinquency? A Role of Microglia in Inflammatory Neurodegenerative Diseases of the CNS

Previous studies have shown that cGMP-dependent protein kinase (PKG) act on several targets in the contractile pathway to reduce intracellular Ca²⁺ and/or augment RhoA-regulated myosin light chain phosphatase (MLCP) activity and cause muscle relaxation. Recent studies have identified a novel protein M-RIP that associates with MYPT1, the regulatory subunit of MLCP. Herein, we examine whether PKG enhance MLCP activity downstream of Ca²⁺ and RhoA via phosphorylation of M-RIP in gastric smooth muscle cells. Treatment of permeabilized muscle cells with 10 μM Ca²⁺ caused an increase in MLC₂₀ phosphorylation and muscle contraction, but had no effect on Rho kinase activity. Activators of PKG (GSNO or cGMP) decreased MLC₂₀ phosphorylation and contraction in response to 10 μM Ca²⁺, implying existence of inhibitory mechanism independent of Ca²⁺ and RhoA. The effect of PKG on Ca²⁺-induced MLC₂₀ phosphorylation was attenuated by M-RIP siRNA. Both GSNO and 8-pCPT-cGMP induced phosphorylation of M-RIP; phosphorylation was accompanied by an increase in the association of M-RIP with MYPT1 and MLCP activity. Taken together, these results provide evidence that PKG induces phosphorylation of M-RIP and enhances its association with MYPT1 to augment MLCP activity and MLC₂₀ dephosphorylation and inhibits muscle contraction, downstream of Ca²⁺- or RhoA-dependent pathways.     

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.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.