Autophosphorylation of smooth-muscle caldesmon.
Caldesmon, a major actin- and calmodulin-binding protein of smooth muscle, has been implicated in regulation of the contractile state of smooth muscle. The isolated protein can be phosphorylated by a co-purifying Ca2+/calmodulin-dependent protein kinase, and phosphorylation blocks inhibition of the actomyosin ATPase by caldesmon [Ngai & Walsh (1987) Biochem. J. 244, 417-425]. We have examined the phosphorylation of caldesmon in more detail. Several lines of evidence indicate that caldesmon itself is a kinase and the reaction is an intermolecular autophosphorylation: (1) caldesmon (141 kDa) and a 93 kDa proteolytic fragment of caldesmon can be separated by ion-exchange chromatography: both retain caldesmon kinase activity, which is Ca2+/calmodulin-dependent; (2) chymotryptic digestion of caldesmon generates a Ca2+/calmodulin-independent form of caldesmon kinase; (3) caldesmon purified to electrophoretic homogeneity retains caldesmon kinase activity, and elution of enzymic activity from a fast-performance-liquid-chromatography ion-exchange column correlates with caldesmon of Mr 141,000; (4) caldesmon is photoaffinity-labelled with 8-azido-[alpha-32P]ATP; labelling is inhibited by ATP, GTP and CTP, indicating a lack of nucleotide specificity; (5) caldesmon binds tightly to Affi-Gel Blue resin, which recognizes proteins having a dinucleotide fold. Autophosphorylation of caldesmon occurs predominantly on serine residues (83.3%), with some threonine (16.7%) and no tyrosine phosphorylation. Autophosphorylation is site-specific: 98% of the phosphate incorporated is recovered in a 26 kDa chymotryptic peptide. Complete tryptic/chymotryptic digestion of this phosphopeptide followed by h.p.l.c. indicates three major phosphorylation sites. Caldesmon exhibits a high degree of substrate specificity: apart from autophosphorylation, brain synapsin I is the only good substrate among many potential substrates examined. These observations indicate that caldesmon may regulate its own function (inhibition of the actomyosin ATPase) by Ca2+/calmodulin-dependent autophosphorylation. Furthermore, caldesmon may regulate other cellular processes, e.g. neurotransmitter release, through the Ca2+/calmodulin-dependent phosphorylation of other proteins such as synapsin I.
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
G C
GC
Scott-Woo
M P
MP
Walsh
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D002918:Chymotrypsin; D007122:Immunoelectrophoresis; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification; D013379:Substrate Specificity
D000345:Affinity Labels; D001386:Azides; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C056959:caldesmon kinase; D002918:Chymotrypsin