Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase.

Huang, Kuo‐Ping; Chan, K F; Singh, Tejinder; Nakabayashi, Hiroki; Huang, Freesia L.

doi:10.1016/s0021-9258(18)67213-8

Cited by 185 publications

(20 citation statements)

References 48 publications

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“…Moreover, Fig. 9 shows the same peptides as those reported for a tryptic digest of protein kinase C from brain (10). Clearly the 80 kD protein substrate is the enzyme itself which catalyzes classical autophosphorylation; this is a well-recognized property of protein kinase C (13,18,39).…”

Section: Discussionmentioning

confidence: 57%

Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

Papadopoulos

Hall

1989

The Journal of Cell Biology

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Abstract. The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipiddependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca 2. and phospholipid, stimulation by tumor promoters but not by nontumorpromoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33-and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [3,-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca 2÷, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP. p ROTEIN kinase C is known to occur in at least three types of adrenocortical cells (35,36). Attempts to determine whether or not this enzyme is involved in the regulation of steroid synthesis in these cells have given equivocal results (36). Since the cytoskeleton of adrenal cells is involved in the steroidogenic responses to ACTH and cyclic AMP (8,22,27), it was decided to determine whether protein kinase C is present in the cytoskeletons of adrenocortical cells. For this purpose we have used Y-1 mouse adrenal tumor cells which serve as a useful system for studies of the regulation of adrenal steroidogenesis (16). We show here that protein kinase C is present in the cytoskeletons of these cells. Materials and Methods Preparation and Culture of Cells Preparation of CytoskeletonCells were scraped from the dishes into buffer A: NaCI (100 mM), sucrose (300 mM), MgCI2 (3 mM), Pipes (10 raM, pH 6.8), Triton X-100 (0.5%), PMSF (1.2 mM), and aprotinin (Trasylol; 100 U/ml). Pancreatic deoxyribonuclease I (400/~g/ml) and pancreatic ribonuclease A (400 #g/ml) were then added for 15 min at room temperature. (NH4)2SO4 was added to a final concentration of 0.1 M and ...

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Section: Discussionmentioning

confidence: 57%

Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

Papadopoulos

Hall

1989

The Journal of Cell Biology

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“…All of our attempts to pr ovid e support for th e idea th a t fasein is ph osphoryla ted hav e been nega tive. These efforts included : (i) treatmen t with acid or alka line phosphatase, which did not chan ge the t wo-di me nsio nal gel pattern; (ii) metab olic lab eling of 3T3 fibr obla sts with 32P0 4, which gav e no incorp oration of label into fasein ; (iii) ph osphorylation trials using purified rat brain pr otein kin ase C (Huang et al, 1986), which failed to lab el r-fa sein ; a nd (iv) a mino a cid a na lysis of r-fa scin , wh ich indicated that only trace amounts of phosphoamino acids were present. Th e results of these experime nts (data not shown) sugges t that fascin isoform s a re not t he resul t of phosphorylation.…”

Section: Resultsmentioning

confidence: 99%

Cloning and Expression of a Murine Fascin Homolog from Mouse Brain

Edwards

Herrera-Sosa

Otto

et al. 1995

Journal of Biological Chemistry

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The fascins are a widely distributed family of proteins that organize filamentous actin into bundles. We have cloned, sequenced, and expressed the murine homolog. Fascin is most abundant in brain and is found in other tissues including uterus and spleen. The deduced open reading frame encodes a protein of 493 amino acids with a molecular mass of 54,412 Da. Previous solubility problems with bacterially expressed fascins were overcome by producing the mouse protein as a fusion with Escherichia coli thioredoxin. A method for cleaving the fusion protein and for purifying active recombinant fascin is described. The N-terminal sequence and molecular mass estimated on SDS gels indicate that recombinant fascin is full-length. Two-dimensional gel electrophoresis suggests that recombinant fascin is post-translationally modified in a manner similar to that observed in mouse brain. Recombinant fascin and the fusion protein are recognized by monoclonal anti-fascin antibodies and will bundle rabbit skeletal muscle F-actin in vitro at a stoichiometry of 4.1:1 actin to fascin. Electron cryomicroscopy images show that the reconstituted bundles are highly ordered. However, their fine structure differs from that of echinoid fascin-actin bundles. This structural difference can be attributed to fascin.

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“…This was not the case for the Mr 39 000 peptide, possibly due to its chemical nature. Labeled peptide fragments of the same size have been recovered from rat brain PKC after autophosphorylation and were ascribed to the N and C termini of PKC (Huang et al, 1986b).…”

Section: Resultsmentioning

confidence: 99%

“…It is noteworthy that types Ila and II/? of rat brain PKC are phosphorylated on their serine and threonine residues, whereas types I and III are phosphorylated on their serine residues only (Huang et al, 1986b). In brief, although the single isoform of bovine neutrophil PKC has the same chromatographic behavior as type I of rat brain PKC or bovine brain PKC, it differs from them by the additional phosphorylation of threonine residues; in that, it would resemble more type Ila and II/?…”

Section: Discussionmentioning

confidence: 97%

Purification and characterization of an isoform of protein kinase C from bovine neutrophils

Dianoux¹,

Stasia²,

Vignais³

1989

Biochemistry

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Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase.

Cited by 185 publications

References 48 publications

Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

Cloning and Expression of a Murine Fascin Homolog from Mouse Brain

Purification and characterization of an isoform of protein kinase C from bovine neutrophils

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