scholarly journals Calmodulin-dependent protein kinase II, and not protein kinase C, is sufficient for triggering cell-cycle resumption in mammalian eggs

2005 ◽  
Vol 118 (17) ◽  
pp. 3849-3859 ◽  
Author(s):  
S. Madgwick
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Mammalian Eggs

scholarly journals Phosphorylation of the C-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II

1992 ◽  
Vol 282 (1) ◽  
pp. 139-145 ◽  
Author(s):  
L Fliegel ◽  
M P Walsh ◽  
D Singh ◽  
C Wong ◽  
A Barr
Keyword(s):  
Protein Kinase C ◽  
Amino Acid ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Dependent Protein Kinase ◽  
Terminal Domain ◽  
Kinase C ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Beta Galactosidase

The Na+/H+ exchanger is a pH-regulatory protein that extrudes one H+ ion in exchange for one Na+ ion when intracellular pH declines. A number of studies have shown phorbol ester stimulation of activity in intact cells, leading to the idea that the exchanger is regulated by protein kinase C-mediated phosphorylation in vivo. cDNA encoding the protein has been cloned, and a recent model suggests a large internal cytoplasmic C-terminal domain that may be a site of regulation of the exchanger [Sardet, Franchi & Pouyssegur (1989) Cell 56, 271-280]. We examined this region of the protein using a rabbit cardiac Na+/H+ exchanger cDNA clone. cDNA of the Na+/H+ exchanger, coding for the C-terminal 178 amino acid residues, was cloned into the expression vector pEX-1 and expressed as a fusion protein with beta-galactosidase. The fusion protein reacted with an antibody produced against a synthetic peptide of the C-terminal 13 amino acid residues of the Na+/H+ exchanger, confirming the identity of the expressed protein. Control and experimental pEX-1-Na+/H+ exchanger protein was purified on a p-aminophenyl beta-D-thiogalactopyranoside-agarose column. Purified Ca2+/calmodulin-dependent protein kinase II readily phosphorylated the Na+/H+ exchanger protein in a Ca(2+)- and calmodulin-dependent manner in vitro, but this region of the protein was not a substrate for purified protein kinase C or for the catalytic subunit of cyclic AMP-dependent protein kinase. Control-expressed beta-galactosidase was phosphorylated to a maximal level of 0.77 +/- 0.17 mol of Pi/mol (mean +/- S.E.M., n = 6) whereas the fusion protein was phosphorylated to a maximal level of 4.09 +/- 0.39 mol of Pi/mol (n = 6), suggesting one site of phosphorylation in beta-galactosidase and three in the C-terminal domain of the Na+/H+ exchanger. Examination of the deduced amino acid sequence of this part of the exchanger reveals three consensus sequences for Ca2+/calmodulin-dependent protein kinase II. These results suggest that the exchanger may be directly regulated in vivo by calmodulin-dependent protein kinase II but not by protein kinase C or cyclic AMP-dependent protein kinase.

scholarly journals Calponin phosphorylation in vitro and in intact muscle

1993 ◽  
Vol 296 (3) ◽  
pp. 827-836 ◽  
Author(s):  
S J Winder ◽  
B G Allen ◽  
E D Fraser ◽  
H M Kang ◽  
G J Kargacin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Gel Electrophoresis ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Sds Page ◽  
Protein Kinase Ii ◽  
Dependent Protein

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.

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