Smooth-muscle mitogen-activated protein (MAP) kinase: purification and characterization, and the phosphorylation of caldesmon.
A single 42 kDa isoform of mitogen-activated protein (MAP) kinase is expressed in both embryonic and adult chicken gizzard. The gizzard MAP kinase, which cross-reacts with anti-p44mpk antibody, has been purified from adult chicken gizzard and partially characterized. The purification protocol employs phenyl-Sepharose, polylysine-agarose, hydroxyapatite, Mono-Q and phenyl-Superose column chromatography. The purified enzyme phosphorylates myelin basic protein and gizzard high-molecular-mass (h-)caldesmon. Sea-star p44mpk and gizzard MAP kinase phosphorylate h-caldesmon at identical sites at the C-terminal domain, as revealed by tryptic-peptide mapping of the phosphorylated protein. Phosphorylation of h-caldesmon by gizzard MAP kinase abolishes its interaction with polymerized tubulin. The specific activity of the purified gizzard kinase toward myelin basic protein is similar to that of brain tau kinase, but is only a fraction of that of activated sea-star p44mpk. This suggests that, although a large amount of MAP kinase is present in the gizzard, only a small percentage of the enzyme is activated normally. Autophosphorylation of the gizzard kinase, at least in part on tyrosine residues, activates its kinase activity.
Childs
T J
TJ
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Mak
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002148:Calmodulin-Binding Proteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1