[37] Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle

Phospholamban is the regulator of the Ca2+-ATPase in cardiac sarcoplasmic reticulum (SR). It is phosphorylated by a Ca2+lcalmodulin-dependeut protein kinase (SRCaM kinase) which is closely associated with cardiac SR membrane preparations. We found that, upon renaturation of pig cardiac SR proteins, blotted onto PVDF membrane, two polypeptides of 54 and 52 kDa showed Ca2+lcalmodulin-dependent autophosphorylation. In Western blots of SR proteins, the 54/52 kDa polypeptides were recognized by an antibody specific for the &CaM kinase isoforms, but not by an anti-a-CaM kinase. The two polypeptides were selectively immunoprecipitated from solubilized SR vesicles with the anti-&CaM kinase. The CaM kinase inhibitors KN-62 and peptide CaMK-(281-302) inhibited the activity of the SRCaM kinase with IC5o values in the same range with those obtained for the brain isozyme. In addition, initial autophosphorylation (Ca2+-dependent) produced a partially Ca2+-independent enzyme while further autophosphorylation (CaZ+-independent) made the enzyme completely Ca2+-independent. Based on these results we suggest that the SRCaM kinase is a distinct &CaM kinase isozyme.

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“…3000 Ci/mmol), Amersham; Protein Phosphatase-2A was prepared according to [14]. Calmodulin prepared according to [15] was a gift from Dr.…”

Section: Methodsmentioning

confidence: 99%

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ‐CaM kinase isozyme

1995

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“…The catalytic subunits of PPl and PP2A [18], and inhibitors-l and -2 [IS] were purified to homogeneity from rabbit skeletal muscle; okadaic acid was a gift from Dr Y. Tsukitani, Fujisawa Pharmaceutical Company, Japan, and microcystin-LR was provided by Professor G. Codd, Department of Biological Sciences, University of Dundee. PDIO, Mono-Q and Superose-12 columns were from Pharmacia/LKB, Freiburg.…”

Section: Methodsmentioning

confidence: 99%

“…PPl and PPM were assayed in the absence of divalent cations using 10 PM "P-1abelled glycogen phosphorylase [18]. When assaying extracts, PPl was the phosphorylase phosphatase activity not inhibited by 1 nM okadaic acid and inhibited by inhibitor-t, while PPZA was the activity inhibited by 1 nM okadaic acid and resistant to inhibitor-2 [19].…”

Section: Protein Phosphatase Assaysmentioning

confidence: 99%

“…by incubation with mammalian protein phosphatases Partially-purified SPS contained no detectable contaminating protein phosphatase activity, because PPl and PP2A both bind to amino-hexyl groups at salt concentrations where SPS is eluted (Methods 2.5., [18]). Incubation of low-activity SPS with 60 mU -ml-' PP2A caused a small increase in maximal activity and a 3-fold increase in activity measured in the presence of 2.5 mM Pi (Table I).…”

Section: Activation Of Partially Purified Low-activity Spsmentioning

confidence: 99%

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Sucrose‐phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves

1990

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Sucrose-phosphate synthase (SPS) purified from spinach leaves harvested in the dark, was activated by mammalian protein phosphatase 2A (PPZA). Activation of SPS in a fraction from darkened spinach leaves was largely prevented by either okadaic acid or microcystin-LR (specific inhibitors of PPI and PP2A), while inhibitor-2 (a PPl inhibitor) or Mg2' (essential for PP2C) were ineffective. In vivo, okadaic acid and microcystin-LR prevented the light-induced activation of SPS and decreased sucrose biosynthesis and CO* fixation. It is concluded that PPZA is the major SPS phosphatase in spinach. This study is the first to employ microcystin-LR for modulating protein phosphorylation in vivo.

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“…"P-Labelled rabbit skeletal muscle phosphorylase a (containing 1.0 mol phosphate per mol subunit) was prepared by phosphorylation with phosphorylase kinase [30], and thiophosphorylase a was obtained in an identical manner except that unlabelled adenosine 5'-[ythioltriphosphate replaced [y-"P]ATP. "P-Labeled rabbit skeletal muscle glycogen synthase was phosphorylated in the site-3 region to 1.5 mollmol subunit with glycogen synthase kinase-3 [31].…”

Section: Preparation Of Phosphorylated Proteins and Phosphatase Assaysmentioning

confidence: 99%

Amino acid sequence and expression of the hepatic glycogen‐binding (G_L‐subunit of protein phosphatase‐1

et al. 1995

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A full-length cDNA encoding the putative hepatic glycogen-binding (G,) subunit of protein phosphatase-1 (PPl) was isolated from a rat liver library. The deduced amino acid sequence (284 residues, 32.6 kDa) was 23% identical (39% similar) to the N-terminal region of the glycogen-binding (G,) subunit of PPl from striated muscle. The similarities between GM and G, were most striking between residues 63-f&& 16166 and 186-227 of human G, (-40% identity), nearly all the identities with the putative yeast homologue GACl being located between 144-166 and 186-227. The cDNA was expressed in E. coli, and the expressed protein transformed the properties of PPl to those characteristic of the hepatic glycogen-associated enzyme. These experiments establish that the cloned protein is G,.

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[37] Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle

Cited by 424 publications

References 16 publications

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ‐CaM kinase isozyme

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ‐CaM kinase isozyme

Sucrose‐phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves

Amino acid sequence and expression of the hepatic glycogen‐binding (G_L‐subunit of protein phosphatase‐1

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[37] Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle

Cited by 424 publications

References 16 publications

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ‐CaM kinase isozyme

The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct δ‐CaM kinase isozyme

Sucrose‐phosphate synthase is dephosphorylated by protein phosphatase 2A in spinach leaves

Amino acid sequence and expression of the hepatic glycogen‐binding (GL‐subunit of protein phosphatase‐1

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Amino acid sequence and expression of the hepatic glycogen‐binding (G_L‐subunit of protein phosphatase‐1