scholarly journals Phosphorylation of pig brain microtubule proteins. General properties and partial characterization of endogenous substrate and cyclic AMP-dependent protein kinase

1977 ◽  
Vol 168 (3) ◽  
pp. 533-539 ◽  
Author(s):  
P Sheterline
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Kinase Activity ◽  
Inhibitor Protein ◽  
Endogenous Protein ◽  
Endogenous Substrate

1. A simple purification procedure for microtubule proteins is described, which involves a single assembly step in vitro in the absence of glycerol, followed by centrifugation through sucrose. 2. The preparation contains 80% tubulin (mol.wt. 54000), 15-20% of a 280000-mol.wt. protein and several other minor components of intermediate molecular weight after polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. 3. In the presence of [gamma-32P]ATP, [32P]phosphate was incorporated into the 280000-mol.wt. component reaching half-maximal incorporation at 1-2 min, but no phosphorylation of tubulin was detected. Cyclic AMP (Km 0.8 micrometer) increased both the initial rate and the extent of incorporation of [32P]phosphate into this component. 4. About half of the endogenous protein kinase activity did not require cyclic AMP and was not inhibited by a heat-stable inhibitor protein from muscle. The remainder of the activity was cyclic AMP-dependent and sensitive to the inhibitor protein. A regulatory subunit was not dissociable from microtubules assembled in vitro in the presence of saturating concentrations of cyclic AMP. 5. The endogenous substrate and the endogenous protein kinase activity could be partially resolved chromatography on phosphocellulose. 6. The data show that cyclic AMP can moduate the activity of an endogenous protein kinase(s) with unusual properties and which phosphorylates a prominent microtubule-associated protein.

scholarly journals The Archaeon Sulfolobus solfataricusContains a Membrane-Associated Protein Kinase Activity That Preferentially Phosphorylates Threonine Residues In Vitro

2000 ◽  
Vol 182 (12) ◽  
pp. 3452-3459 ◽  
Author(s):  
Brian H. Lower ◽  
Kenneth M. Bischoff ◽  
Peter J. Kennelly
Keyword(s):  
Protein Kinase ◽  
Molecular Mass ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Kinase Activity ◽  
Histone H4 ◽  
Purine Nucleotides ◽  
Archaeal Protein

ABSTRACT The extreme acidothermophilic archaeon Sulfolobus solfataricus harbors a membrane-associated protein kinase activity. Its solubilization and stabilization required detergents, suggesting that this activity resides within an integral membrane protein. The archaeal protein kinase utilized purine nucleotides as phosphoryl donors in vitro. A noticeable preference for nucleotide triphosphates over nucleotide diphosphates and for adenyl nucleotides over the corresponding guanyl ones was observed. The molecular mass of the solubilized, partially purified enzyme was estimated to be ≈125 kDa by gel filtration chromatography. Catalytic activity resided in a polypeptide with an apparent molecular mass of ≈67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Challenges with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, histone H4, reduced carboxyamidomethylated and maleylated lysozyme, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels in vitro. All of the aforementioned substrates were phosphorylated on threonine residues, while histone H4 was phosphorylated on serine as well. Substitution of serine for the phosphoacceptor threonine in the myosin light chain peptide produced a noticeably inferior substrate. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to a set of known inhibitors of “eukaryotic” protein kinases.

Stimulation of calcium accumulation in cardiac sarcolemma by protein kinase

1976 ◽  
Vol 54 (5) ◽  
pp. 438-445 ◽  
Author(s):  
Prakash V. Sulakhe ◽  
Nicholas Ling-Kit Leung ◽  
Patrick J. St. Louis
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Cardiac Sarcolemma ◽  
Guinea Pig Heart ◽  
Calcium Accumulation ◽  
Exogenous Protein ◽  
Endogenous Protein ◽  
Membrane Phosphorylation

Sarcolemmal membranes isolated from guinea pig heart ventricles contained an ATP-dependent calcium-sequestering activity. Sarcolemmai calcium accumulation but not binding was enhanced by preincubation of membranes with exogenous protein kinase, with cyclic AMP, or with isoproterenol. Protein kinase (EC 2.7.1.37) increased the V of Ca2+ accumulation by sarcolemma without any significant effect on the affinity for Ca2+. The endogenous protein kinase activity present in isolated sarcolemma affected membrane phosphorylation. Cyclic AMP increased the endogenous kinase activity modestly, whereas histone increased it significantly. Exogenous protein kinase also catalyzed phosphorylation of these membranes. Endogenous and exogenous kinase-catalyzed phosphorylation of sarcolemma was hydroxylamine-insensitive. Ca2+-dependent ATPase (EC 3.6.1.3) (extra ATPase) activity of sarcolemma was also increased by protein kinase.

scholarly journals Comparison of Ca2+-dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain

1985 ◽  
Vol 232 (3) ◽  
pp. 629-635 ◽  
Author(s):  
L L Norling ◽  
M Landt
Keyword(s):  
Protein Kinase ◽  
Gel Electrophoresis ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Cells ◽  
Synapsin I ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.

scholarly journals A phospholipid-stimulated protein kinase from Dictyostelium discoideum

1989 ◽  
Vol 260 (2) ◽  
pp. 557-561 ◽  
Author(s):  
B Jiménez ◽  
A Pestaña ◽  
M Fernandez-Renart
Keyword(s):  
Protein Kinase ◽  
Protease Inhibitors ◽  
Dictyostelium Discoideum ◽  
Kinase Activity ◽  
Deae Cellulose ◽  
Protein Kinase Activity ◽  
Endogenous Protein ◽  
Exogenous Substrate ◽  
Intermolecular Reactions

A protein kinase with unusual characteristics has been found in Dictyostelium discoideum. This kinase can use histone H1 as exogenous substrate, and the activity is stimulated by phospholipids, but not by Ca2+. This enzyme has been partially purified by using chromatography on DEAE-cellulose DE-52, spermine-agarose and phosphatidylserine-polyacrylamide. The protein kinase activity is very labile, even in the presence of protease inhibitors, making further purification difficult. In the activity-containing fractions, an endogenous protein of 140 kDa is labelled in vitro with [gamma-32P]ATP under conditions in which intramolecular rather than intermolecular reactions are favoured. This protein is labelled only in the presence of phospholipids, but not of Ca2+. We propose that the 140 kDa phosphoprotein might be the autophosphorylated enzyme.

scholarly journals Highly Purified pp60srcInduces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

1983 ◽  
Vol 3 (1) ◽  
pp. 102-112 ◽  
Author(s):  
Patricia F. Maness ◽  
Barcey T. Levy
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Sodium Dodecyl ◽  
Active Agent ◽  
Cytoskeletal Proteins ◽  
Protein Kinase Activity ◽  
Tumor Bearing ◽  
Rabbit Igg ◽  
Associated Proteins

Thesrcgene product of Rous sarcoma virus (pp60src) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60srcfraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60srcby immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60src. A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60srcwas achieved by this procedure. Purified pp60srcphosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [γ-32P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60srcbecame labeled with32P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60srcinto the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60src, thus implicating pp60srcas the active agent. Examination of actin-associated proteins as substrates for the pp60srckinase in vitro showed that vinculin was phosphorylated directly by pp60src, although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60srcpurified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.

Highly Purified pp60 src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

1983 ◽  
Vol 3 (1) ◽  
pp. 102-112
Author(s):  
Patricia F. Maness ◽  
Barcey T. Levy
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Sodium Dodecyl ◽  
Active Agent ◽  
Cytoskeletal Proteins ◽  
Protein Kinase Activity ◽  
Tumor Bearing ◽  
Rabbit Igg ◽  
Associated Proteins

The src gene product of Rous sarcoma virus (pp60 src ) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60 src fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60 src by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60 src . A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60 src was achieved by this procedure. Purified pp60 src phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [γ- 32 P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60 src became labeled with 32 P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60 src into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60 src , thus implicating pp60 src as the active agent. Examination of actin-associated proteins as substrates for the pp60 src kinase in vitro showed that vinculin was phosphorylated directly by pp60 src , although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60 src purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.

scholarly journals Modulation of testicular galactolipid sulphotransferase activity by phosphorylation. Stimulation of enzyme activity in vitro by an endogenous kinase

1989 ◽  
Vol 261 (2) ◽  
pp. 423-429 ◽  
Author(s):  
D Sakac ◽  
C A Lingwood
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Affinity Chromatography ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
General Method

Evidence is presented for a testicular protein kinase activity capable of stimulating the activity in vitro of a partially purified preparation of the testicular galactolipid sulphotransferase. This enzyme is responsible for the synthesis of the major mammalian testicular glycolipid, sulphogalactosylglycerol, and is an early marker of differentiation during spermatogenesis. This stimulatory activity has been separated by affinity chromatography, using 3′,5′-ADP-agarose, from both the detergent-solubilized microsomes (microsomal fractions) and the soluble fraction of the testicular homogenate. The stimulator was eluted from the affinity matrix by either a salt, or, more selectively, a cyclic AMP gradient. Thus this matrix can function as an analogue of 3′,5′-cyclic AMP. The activity of the sulphotransferase stimulator was ATP-dependent and coincident with protein kinase activity. Sulphotransferase activity was also stimulated in the presence of commercial preparations of cyclic AMP-dependent protein kinase and stimulation was prevented in the presence of kinase inhibitors. Our results suggest that sulphogalactolipid biosynthesis is regulated by a phosphorylation process during spermatogenesis. In addition, our results suggest that affinity chromatography on 3′,5′-ADP-agarose may provide a general method for the purification of cyclic AMP-dependent kinases.

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