Sutherland C, Renaux B S, McKay D J, Walsh M P
MRC Group in Signal Transduction, University of Calgary, Alberta, Canada.
J Muscle Res Cell Motil. 1994 Aug;15(4):440-56. doi: 10.1007/BF00122118.
A caldesmon kinase activity was partially purified from an extract of chicken gizzard smooth muscle by sequential chromatography on columns of DEAE-Sephacel, MonoQ and Superose 12. This kinase was identified as casein kinase II by Western blotting using peptide-directed antibodies raised against the alpha, alpha' and beta subunits of human casein kinase II; the smooth muscle enzyme consisted of similar subunits of M(r) 43,000 (alpha), 39,000 (alpha'), and 27,000 (beta). Phosphorylation of caldesmon and casein by smooth muscle casein kinase II was optimal at approximately 0.1 M NaCl, did not require second messengers, and was inhibited by heparin. The kinase utilized either GTP or ATP as a substrate. Caldesmon was phosphorylated to approximately 1 mol Pi mol-1 caldesmon by smooth muscle casein kinase II with a Km for caldesmon of 4.9 microM. Two-dimensional thin-layer electrophoresis indicated phosphate incorporation into both serine and threonine. All the incorporated phosphate was recovered in the N-terminal peptide (residues 1-152) generated by cleavage at cysteine 153 with 2-nitro-5-thiocyanobenzoic acid. Purification of tryptic phosphopeptides and N-terminal sequencing revealed two principal sites of phosphorylation: serine 73 and threonine 83. The following four synthetic peptides corresponding to this domain of caldesmon were examined as substrates of casein kinase II: A = RRREVNAQNSVAEEE; B = AQNSVAEEE; C = RSTDDEAA; D = SVAEEETKRSTDDE. Interestingly, only peptides C and D were phosphorylated and both only at threonine. Phosphorylation of intact caldesmon did not affect the pattern of chymotryptic digestion suggesting that it does not induce a significant conformational change in the protein substrate. Phosphorylation also had no effect on the binding of caldesmon to actin or on the caldesmon-mediated inhibition of actomyosin MgATPase activity. However, phosphorylation completely abolished the interaction of caldesmon with immobilized smooth muscle myosin. These results are consistent with the localization of the myosin-binding domain near the N-terminus of caldesmon and of the actin-binding domain near the opposite end of the elongated molecule. Casein kinase II may therefore play a role in regulating caldesmon-myosin interaction and the ability of caldesmon to cross-link actin and myosin filaments in smooth muscle.
通过在DEAE - Sephacel、MonoQ和Superose 12柱上的连续层析，从鸡砂囊平滑肌提取物中部分纯化了一种钙调蛋白激酶活性。使用针对人酪蛋白激酶II的α、α'和β亚基产生的肽导向抗体，通过蛋白质印迹法将该激酶鉴定为酪蛋白激酶II；平滑肌酶由分子量为43,000（α）、39,000（α'）和27,000（β）的相似亚基组成。平滑肌酪蛋白激酶II对钙调蛋白和酪蛋白的磷酸化在约0.1 M NaCl时最适宜，不需要第二信使，并被肝素抑制。该激酶可利用GTP或ATP作为底物。平滑肌酪蛋白激酶II将钙调蛋白磷酸化至约1 mol Pi/mol - 1钙调蛋白，钙调蛋白的Km为4.9 μM。二维薄层电泳表明磷酸盐掺入丝氨酸和苏氨酸。所有掺入的磷酸盐都在通过用2 - 硝基 - 5 - 硫氰基苯甲酸在半胱氨酸153处切割产生的N端肽（残基1 - 152）中回收。胰蛋白酶磷酸肽的纯化和N端测序揭示了两个主要的磷酸化位点：丝氨酸73和苏氨酸83。检查了与钙调蛋白该结构域相对应的以下四个合成肽作为酪蛋白激酶II的底物：A = RRREVNAQNSVAEEE；B = AQNSVAEEE；C = RSTDDEAA；D = SVAEEETKRSTDDE。有趣的是，只有肽C和D被磷酸化，且都仅在苏氨酸处。完整钙调蛋白的磷酸化不影响胰凝乳蛋白酶消化模式，表明它不会在蛋白质底物中诱导显著的构象变化。磷酸化对钙调蛋白与肌动蛋白的结合或对钙调蛋白介导的肌动球蛋白MgATPase活性的抑制也没有影响。然而，磷酸化完全消除了钙调蛋白与固定化平滑肌肌球蛋白的相互作用。这些结果与肌球蛋白结合结构域在钙调蛋白N端附近以及肌动蛋白结合结构域在细长分子另一端附近的定位一致。因此，酪蛋白激酶II可能在调节平滑肌中钙调蛋白 - 肌球蛋白相互作用以及钙调蛋白交联肌动蛋白和肌球蛋白丝的能力方面发挥作用。
