Identification and characterization of cAMP-dependent protein kinase and its possible direct interactions with protein phosphatase-1 in marine dinoflagellates

1996 ◽  
Vol 74 (4) ◽  
pp. 559-567 ◽  
Author(s):  
John F. Dawson ◽  
Kathy He Wang ◽  
Charles F. B. Holmes
Keyword(s):  
Signal Transduction ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Phosphorylation Site ◽  
Protein Phosphatase 1 ◽  
Dependent Protein Kinase ◽  
Prorocentrum Lima ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

We have examined the nature of signal transduction involving reversible protein phosphorylation in marine Prorocentrale species. Of particular interest is the marine dinoflagellate Prorocentrum lima in which the tumour promoter okadaic acid is produced and may interfere with signal transduction. We have identified cAMP-dependent protein kinase (PKA) activity in P. lima, P. micans, and P. minimum. The P. lima enzyme was characterized biochemically and appears to consist of two different isoforms in the R2C2 configuration. Whole cell extracts of P. micans and P. minimum treated with the specific PKA inhibitor peptide PKI (5–24) or cAMP demonstrated altered intensities of phosphopeptide 32P labeling, most likely involving regulation of a protein phosphatase via PKA activity. A primary candidate for PKA regulation is protein phosphatase-1 (PP-1), which in P. lima possesses a classical PKA consensus phosphorylation site. We demonstrate that a peptide fragment of PP-1 from P. lima corresponding to this PKA phosphorylation site can be effectively phosphorylated by PKA and dephosphorylated by calcineurin. We speculate that PP-1 activity among several lower eukaryotes may be mediated directly by reversible phosphorylation. Higher eukaryotes may have developed inhibitor proteins to provide more complex regulation of protein phosphatase activity.Key words: cAMP-dependent protein kinase, protein phosphatase-1, dinoflagellates, Prorocentrum lima, okadaic acid.

Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells

1992 ◽  
Vol 262 (4) ◽  
pp. G763-G773 ◽  
Author(s):  
J. R. Goldenring ◽  
V. A. Asher ◽  
M. F. Barreuther ◽  
J. J. Lewis ◽  
S. M. Lohmann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Parietal Cells ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Endogenous Proteins

The phosphorylation of endogenous proteins was investigated in subcellular fractions prepared from isolated rabbit parietal cells incubated with either cimetidine (unstimulated) or a combination of histamine and forskolin (maximally stimulated). Phosphorylation of endogenous proteins in subfractions was then assessed in a post hoc assay using [gamma-32P]ATP as a phosphate donor in vitro. The Mg(2+)-dependent incorporation of [32P]phosphate into a 52-kDa protein (pp52M) was observed in the 4,000 g membrane fraction from stimulated but not unstimulated cells. The pp52M protein was identified as the type II regulatory subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (RII) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [32P]phosphate into RII in the in vitro assay in the presence of Zn2+ was apparent in the 4,000 g membrane from stimulated but not unstimulated cells. The results thus suggested that, on stimulation, RII in membrane was dephosphorylated. Incorporation of [32P]phosphate into membrane-associated RII was completely abolished in the presence of 10 microM cAMP. The decrease in RII phosphorylation in membrane from stimulated cells assayed in the presence of cAMP was due to a phosphoprotein phosphatase activity that was completely inhibited by okadaic acid (1 microM). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound RII by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [14C]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.

scholarly journals LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

2000 ◽  
Vol 345 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Sean P. COLLINS ◽  
Junewai L. REOMA ◽  
David M. GAMM ◽  
Michael D. UHLER
Keyword(s):  
Protein Kinase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Multiple Cancer ◽  
Dependent Protein Kinase ◽  
Rnase Protection ◽  
Camp Dependent Protein Kinase ◽  
Increased Risk ◽  
Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

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