A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.

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Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins

Blood ◽

10.1182/blood.v75.2.479.479 ◽

1990 ◽

Vol 75 (2) ◽

pp. 479-487 ◽

Cited By ~ 7

Author(s):

SJ Stoehr ◽

JE Smolen

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Neutrophils ◽

Major Protein ◽

Guanosine Triphosphate ◽

Alternative Mode ◽

Kinase C ◽

Phosphate Donor

Abstract Investigations of protein kinase C (PKC) activity have focussed on protein phosphorylation using adenosine triphosphate (ATP), not guanosine triphosphate (GTP), as the phosphate donor. In a continuing study of the enzymology of the PKC of human neutrophils, we wanted to determine if there might be protein kinases that do use GTP as a phosphate donor. Soluble extracts or detergent-extracted fractions of human neutrophils were used as enzyme sources. Phosphorylation of histone using [gamma-32P]-GTP was 31% as effective as [gamma-32P]-ATP. Phosphorylation with GTP depended on Ca2+, Mg2+, and phospholipid, just as the ATP, and the Ca2+ requirements were similar. In all cases, H-7, an inhibitor of ATP-supported PKC activity, blocked GTP-utilizing activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that similar endogenous proteins were phosphorylated with ATP or GTP. The apparent Km and Vmax for the enzyme(s) for both phosphate donors were identical, although these were modified by treatment with Triton X-100. GTP competitively inhibited use of ATP by PKC; however, low concentrations of ATP enhanced GTP- utilizing kinase activity in some cases. Non-hydrolyzable forms of ATP and other nucleotide triphosphates were inhibitory. Detergent treatment also markedly altered the number of proteins phosphorylated by either nucleotide. The major protein phosphorylated in the soluble or detergent extract was a single polypeptide band in the 34 Kd range. These studies are the first to explicitly examine the possible phosphorylation by neutrophil PKC using GTP and point to a potential alternative mode of enzyme activity. Since high concentrations of GTP are available within neutrophils, the ability of PKC or a PKC-like enzyme to use this nucleotide may have important ramifications in signal transduction.

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Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins

Blood ◽

10.1182/blood.v75.2.479.bloodjournal752479 ◽

1990 ◽

Vol 75 (2) ◽

pp. 479-487

Author(s):

SJ Stoehr ◽

JE Smolen

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Neutrophils ◽

Major Protein ◽

Guanosine Triphosphate ◽

Alternative Mode ◽

Kinase C ◽

Phosphate Donor

Investigations of protein kinase C (PKC) activity have focussed on protein phosphorylation using adenosine triphosphate (ATP), not guanosine triphosphate (GTP), as the phosphate donor. In a continuing study of the enzymology of the PKC of human neutrophils, we wanted to determine if there might be protein kinases that do use GTP as a phosphate donor. Soluble extracts or detergent-extracted fractions of human neutrophils were used as enzyme sources. Phosphorylation of histone using [gamma-32P]-GTP was 31% as effective as [gamma-32P]-ATP. Phosphorylation with GTP depended on Ca2+, Mg2+, and phospholipid, just as the ATP, and the Ca2+ requirements were similar. In all cases, H-7, an inhibitor of ATP-supported PKC activity, blocked GTP-utilizing activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that similar endogenous proteins were phosphorylated with ATP or GTP. The apparent Km and Vmax for the enzyme(s) for both phosphate donors were identical, although these were modified by treatment with Triton X-100. GTP competitively inhibited use of ATP by PKC; however, low concentrations of ATP enhanced GTP- utilizing kinase activity in some cases. Non-hydrolyzable forms of ATP and other nucleotide triphosphates were inhibitory. Detergent treatment also markedly altered the number of proteins phosphorylated by either nucleotide. The major protein phosphorylated in the soluble or detergent extract was a single polypeptide band in the 34 Kd range. These studies are the first to explicitly examine the possible phosphorylation by neutrophil PKC using GTP and point to a potential alternative mode of enzyme activity. Since high concentrations of GTP are available within neutrophils, the ability of PKC or a PKC-like enzyme to use this nucleotide may have important ramifications in signal transduction.

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Immunological evidence for two physiological forms of protein kinase C

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.85-96.1987 ◽

1987 ◽

Vol 7 (1) ◽

pp. 85-96

Author(s):

J R Woodgett ◽

T Hunter

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Mammalian Cells ◽

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Enzyme Treatment ◽

One Dimensional ◽

Kinase C ◽

Differential Phosphorylation

Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+.

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Immunological evidence for two physiological forms of protein kinase C.

Molecular and Cellular Biology ◽

10.1128/mcb.7.1.85 ◽

1987 ◽

Vol 7 (1) ◽

pp. 85-96 ◽

Cited By ~ 67

Author(s):

J R Woodgett ◽

T Hunter

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Mammalian Cells ◽

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Enzyme Treatment ◽

One Dimensional ◽

Kinase C ◽

Differential Phosphorylation

Our recently described purification scheme for rat brain protein kinase C yields an enzyme consisting of a 78/80-kilodalton (kDa) doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (submitted for publication). Antisera against this preparation were raised in two rabbits. One of the antisera detected only the 80-kDa component by immunoblotting of purified protein kinase C and immunoprecipitated an 80-kDa [35S]methionine-labeled protein from a variety of human, rodent, and bovine cells, which was shown to represent protein kinase C by comparative one-dimensional peptide mapping. In contrast, the second antiserum detected both 78- and 80-kDa enzyme forms by immunoblotting and immunoprecipitated a [35S]methionine-labeled 78/80-kDa doublet from mammalian cells. One-dimensional peptide maps of these 78- and 80-kDa proteins were similar to those derived from the 78- and 80-kDa forms of purified protein kinase C, respectively. The two forms were not related by either partial proteolysis or differential phosphorylation, showing that two distinct forms of this enzyme exist in mammalian cells. Treatment of mouse B82 L cells with 2.5 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) per ml for 18 h resulted in complete loss of immunoprecipitable protein kinase C with a half time of disappearance of 48 min. Since the normal half-life of protein kinase C was greater than 24 h and the biosynthetic rate of the protein was not decreased after 18 h by TPA treatment, TPA induces down-regulation by increasing the degradation rate of the enzyme. Treatment of cells with 50 ng of TPA per ml followed by resolution of the membrane and cytosol in the presence of ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) promoted an apparent translocation of both 78- and 80-kDa proteins from the cytosol to the membrane fraction. A similar translocation was effected by cell lysis in the presence of Ca2+, indicating the subcellular localization of protein kinase C to be sensitive to the presence of both activators and micromolar amounts of Ca2+.

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Faculty Opinions recommendation of Exercise improves phosphatidylinositol-3,4,5-trisphosphate responsiveness of atypical protein kinase C and interacts with insulin signalling to peptide elongation in human skeletal muscle.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1089955.543122 ◽

2008 ◽

Author(s):

Mark Hargreaves

Keyword(s):

Skeletal Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Human Skeletal Muscle ◽

Insulin Signalling ◽

Atypical Protein Kinase C ◽

Atypical Protein Kinase ◽

Kinase C ◽

Peptide Elongation

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Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)31643-0 ◽

1994 ◽

Vol 269 (51) ◽

pp. 32358-32367

Author(s):

A Toker ◽

M Meyer ◽

K K Reddy ◽

J R Falck ◽

R Aneja ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Family Members ◽

The Novel ◽

Kinase C

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Functional divergence of protein kinase C (PKC) family members. PKC gamma differs from PKC alpha and -beta II and nPKC epsilon in its competence to mediate-12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive transcriptional activation through a TPA-response element.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)52986-0 ◽

1993 ◽

Vol 268 (12) ◽

pp. 9122-9129

Author(s):

A. Hata ◽

Y. Akita ◽

K. Suzuki ◽

S. Ohno

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Transcriptional Activation ◽

Functional Divergence ◽

Family Members ◽

Response Element ◽

Kinase C ◽

Pkc Alpha

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Interplay and Effects of Temporal Changes in the Phosphorylation State of Serine-302, -307, and -318 of Insulin Receptor Substrate-1 on Insulin Action in Skeletal Muscle Cells

Molecular Endocrinology ◽

10.1210/me.2008-0102 ◽

2008 ◽

Vol 22 (12) ◽

pp. 2729-2740 ◽

Cited By ~ 39

Author(s):

Cora Weigert ◽

Matthias Kron ◽

Hubert Kalbacher ◽

Ann Kathrin Pohl ◽

Heike Runge ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Insulin Receptor ◽

Insulin Action ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Insulin Signal ◽

Kinase C ◽

Phosphorylation Pattern

Abstract Transduction of the insulin signal is mediated by multisite Tyr and Ser/Thr phosphorylation of the insulin receptor substrates (IRSs). Previous studies on the function of single-site phosphorylation, particularly phosphorylation of Ser-302, -307, and -318 of IRS-1, showed attenuating as well as enhancing effects on insulin action. In this study we investigated a possible cross talk of these opposedly acting serine residues in insulin-stimulated skeletal muscle cells by monitoring phosphorylation kinetics, and applying loss of function, gain of function, and combination mutants of IRS-1. The phosphorylation at Ser-302 was rapid and transient, followed first by Ser-318 phosphorylation and later by phosphorylation of Ser-307, which remained elevated for 120 min. Mutation of Ser-302 to alanine clearly reduced the subsequent protein kinase C-ζ-mediated Ser-318 phosphorylation. The Ser-307 phosphorylation was independent of Ser-302 and/or Ser-318 phosphorylation status. The functional consequences of these phosphorylation patterns were studied by the expression of IRS-1 mutants. The E302A307E318 mutant simulating the early phosphorylation pattern resulted in a significant increase in Akt and glycogen synthase kinase 3 phosphorylation. Furthermore, glucose uptake was enhanced. Because the down-regulation of the insulin signal was not affected, this phosphorylation pattern seems to be involved in the enhancement but not in the termination of the insulin signal. This enhancing effect was completely absent when Ser-302 was unphosphorylated and Ser-307 was phosphorylated as simulated by the A302E307E318 mutant. Phospho-Ser-318, sequentially phosphorylated at least by protein kinase C-ζ and a mammalian target of rapamycin/raptor-dependent kinase, was part of the positive as well as of the subsequent negative phosphorylation pattern. Thus we conclude that insulin stimulation temporally generates different phosphorylation statuses of the same residues that exert different functions in insulin signaling.

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