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) ML CK by the catalytic subunit of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was studied. In contrast to other smooth muscl e 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. Modif ication of serine residues inhibited the activity of the enzyme. PKC a lso incorporated 2.0-2.1 moles of phosphate per mole psmMLCK under bot h conditions but had no effect on the MLCK activity. Sequential phosph orylation by PKC and PKA incorporated 3.8-4.1 moles phosphate suggesti ng that the amino acid residues modified by the two kinases are differ ent. Phosphoamino acid analysis of the MLCK revealed that PKC phosphor ylated serine and threonine residues. The double reciprocal plots of t he enzyme activity and calmodulin concentrations showed that the V-max of the reaction is not altered by phosphorylation by PKA but the calm odulin concentration require for half-maximal activation is increased about 4-fold. Only 10 out of 17 monoclonal antibodies to various regio ns of the turkey gizzard MLCK cross-reacted with psmMLCK suggesting st ructural differences between these enzymes, Comparison of the deduced amino acid sequence of the cDNA encoding the C-terminal half of the ps mMLCK molecule showed that while cgMLCK and psmMLCK are highly homolog ous, a number of nonconservative substitutions are present, particular ly near the PKA phosphorylation site B (S-828).