Autophosphorylation of smooth-muscle caldesmon

Abstract

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.

Flexation of caldesmon: effect of conformation on the properties of caldesmon

“…Second, CDS can be photoaffinity labelled with 8-azido-(0~p32)ATP (Scott-Woo & Walsh, 1988). Indeed, the presence of ATP dramatically decreased the hairpin form of CDS.…”

Ca2+‐independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments

“…All other reagents were analytical grade or better and were purchased from VWR Canlab (Edmonton, Canada) or Sigma. The following proteins were purified as described previously: chicken gizzard myosin II (Persechini & Hartshorne, 1981), MLCK (Ngai et al 1984), Ca 2+ ‐CaM‐dependent protein kinase II (CaM kinase II) with caldesmon (Scott‐Woo & Walsh, 1988) and CaM (Walsh et al 1984), and rat brain protein kinase C (PKC; a mixture of Ca 2+ ‐dependent α, β and γ isoforms) (Allen et al 1994). LC 20 was prepared from purified myosin isolated from 200 g of chicken gizzard using a modification of the procedure of Hathaway & Haeberle (1983).…”

Structural Analysis of a Calmodulin Variant from Rice

“…Subsequently, 32 P was quantified by Cerenkov counting (41). Calmodulin kinase II (CaMKII) was also purified from chicken gizzards (42,43). 0.2 g/ml CaMKII was preincubated at 30°C with 50 M autocamtide 2 peptide substrate, 50 mM MOPS, pH 7.0, 60 mM KCl, 10 mM MgCl 2 , 0.2 mM excess CaCl 2 , and different forms of OsCaM61 and mCaM for 2 min.…”