Phosphorylation and partial sequence of pregnant sheep myometrium myosin light chain kinase.
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 Ca(2+)-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).
Pato
M D
MD
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Kerc
E
E
Lye
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D008969:Molecular Sequence Data; D009215:Myometrium / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000737:chemistry; D010449:Peptide Mapping; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002147:Calmodulin; D015345:Oligonucleotide Probes; D010769:Phosphothreonine; D010768:Phosphoserine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D002118:Calcium