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Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase

  • Mary D. Pato
  • Ewa Kerc
  • Stephen J. Lye
Part of the Developments in Molecular and Cellular Biochemistry book series (DMCB, volume 15)

Abstract

The function of the uterine smooth muscle in gestation and parturition is affected by a variety of hormones and biomolecules, some of which alter the intracellular levels of cAMP and Ca2+. Since the activity of smooth muscle MLCK has been shown to be modulated by phosphorylation, the effect of this modification of pregnant sheep myometrium (psm) MLCK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscle MLCK reported, PKA incorporates 2.0–2.2 moles phosphate into a mole of psm MLCK both in the presence and absence of Ca2+-calmodulin. Modification of serine residues inhibited the activity of the enzyme. PKC also incorporated 2.0–2.1 moles of phosphate per mole psmMLCK under both conditions but had no effect on the MLCK activity. Sequential phosphorylation by PKC and PKA incorporated 3.8–4.1 moles phosphate suggesting that the amino acid residues modified by the two kinases are different. Phosphoamino acid analysis of the MLCK revealed that PKC phosphorylated serine and threonine residues. The double reciprocal plots of the enzyme activity and calmodulin concentrations showed that the Vmax of the reaction is not altered by phosphorylation by PKA but the calmodulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regions of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting structural differences between these enzymes. Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the psmMLCK molecule showed that while cgMLCK and psmMLCK are highly homologous, a number of nonconservative substitutions are present, particularly near the PKA phosphorylation site B (S828).

Key words

myometrium myosin light chain kinase calmodulin phosphorylation 

Abbreviations

MLCK

myosin light chain kinase

psm

pregnant sheep myometrium

tg

turkey gizzard eg-chicken gizzard;

ru

rabbit uterine

bs

bovine stomach

PKA

catalytic subunit of cAMP-dependent protein kinase

PKC

Ca2+ and phospholipid dependent protein kinase

CaM Kinase II

multifunctional Ca2+ and calmodulin dependent protein kinase

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Copyright information

© Springer Science+Business Media Dordrecht 1995

Authors and Affiliations

  • Mary D. Pato
    • 1
  • Ewa Kerc
    • 1
  • Stephen J. Lye
    • 2
  1. 1.Department of BiochemistryUniversity of SaskatchewanSaskatoonCanada
  2. 2.Division of PerinatologySamuel Lunenfeld Research InstituteTorontoCanada

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