Smith M K, Colbran R J, Soderling T R
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Biol Chem. 1990 Feb 5;265(4):1837-40.
Synthetic peptides corresponding to the autoinhibitory domains of calcium/calmodulin-dependent protein kinase II (CaMK-(281-309)), smooth muscle myosin light chain kinase (MLCK-(480-501)), and protein kinase C (PKC-(19-36)) as well as a peptide derived from the heat-stable inhibitor of cAMP-dependent protein kinase (PKI-tide) were tested for their inhibitory specificities. The inhibitory potencies of the four peptides were determined for each of the four protein kinases using both peptide substrates (at approximate Km concentrations) and protein substrates (at concentrations less than Km). In agreement with previous studies PKI-tide was a specific and potent inhibitor of only cAMP kinase, and none of the other inhibitory peptides gave significant inhibition of cAMP kinase at concentrations of less than 100 microM. With synthetic peptide substrates, PKC-(19-36) strongly inhibited native PKC (IC50 less than 1 microM) but also significantly inhibited autophosphorylated CaMK-II (IC50 = 30 microM) and proteolytically activated MLCK (IC50 = 35 microM). MLCK-(480-501) potently inhibited MLCK (IC50 = 0.25 microM) and also strongly inhibited both PKC and CaMK-II (IC50 = 1.4 and 1.7 microM, respectively). CaMK-(281-309) inhibited autophosphorylated CaMK-II, PKC, and proteolyzed MLCK almost equally (IC50 = 10, 38, and 48 microM, respectively). Qualitatively similar results were obtained with protein substrates. These studies validate the use of PKI-tide as a specific inhibitor of cAMP kinase in intact cell studies and suggest that PKC-(19-36) can also be used but only within a narrow concentration range. However, the autoinhibitory domain peptides from MLCK and CaMK-II are not sufficiently specific to be used in similar investigations.
对与钙/钙调蛋白依赖性蛋白激酶II（CaMK-(281 - 309)）、平滑肌肌球蛋白轻链激酶（MLCK-(480 - 501)）和蛋白激酶C（PKC-(19 - 36)）的自身抑制结构域相对应的合成肽，以及源自环磷酸腺苷依赖性蛋白激酶热稳定抑制剂（PKI-肽）的一种肽进行了抑制特异性测试。使用肽底物（在近似Km浓度下）和蛋白质底物（在小于Km的浓度下）测定了这四种肽对四种蛋白激酶中每一种的抑制效力。与先前的研究一致，PKI-肽是仅对环磷酸腺苷激酶具有特异性且强效的抑制剂，并且在浓度低于100微摩尔时，其他抑制性肽均未对环磷酸腺苷激酶产生显著抑制。对于合成肽底物，PKC-(19 - 36)强烈抑制天然PKC（IC50小于1微摩尔），但也显著抑制自身磷酸化的CaMK-II（IC50 = 30微摩尔）和经蛋白水解激活的MLCK（IC50 = 35微摩尔）。MLCK-(480 - 501)有效抑制MLCK（IC50 = 0.25微摩尔），并且也强烈抑制PKC和CaMK-II（分别为IC50 = 1.4和1.7微摩尔）。CaMK-(281 - 309)几乎同等程度地抑制自身磷酸化的CaMK-II、PKC和经蛋白水解的MLCK（分别为IC50 = 10、38和48微摩尔）。使用蛋白质底物获得了定性相似的结果。这些研究验证了PKI-肽在完整细胞研究中作为环磷酸腺苷激酶特异性抑制剂的用途，并表明PKC-(19 - 36)也可使用，但仅在狭窄的浓度范围内。然而，来自MLCK和CaMK-II的自身抑制结构域肽的特异性不足以用于类似的研究。
