George S E, VanBerkum M F, Ono T, Cook R, Hanley R M, Putkey J A, Means A R
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1990 Jun 5;265(16):9228-35.
To evaluate the role of domain I of calmodulin (CaM) in the activation of target enzymes, a series of CaM mutants was constructed in which domain I (49 amino acids) was substantially deleted, or was exchanged with the homologous region (58 amino acids) of cardiac troponin C (cTnC). The proteins are 1) aM, a mutant CaM in which domain I has been deleted; 2) TaM, first domain of cTnC, last three domains of CaM; 3) TaM-BMI, same as TaM, except the nonfunctional first Ca2(+)-binding domain has been restored by mutagenesis; 4) CaT, first domain of CaM, last three domains of cTnC. These proteins were evaluated for Ca2+ binding properties and as activators of three CaM target enzymes, CaM-dependent phosphodiesterase (PDE), smooth muscle myosin light chain kinase (MLCK), and CaM-dependent multifunctional protein kinase (CaM kinase II). The chimeric proteins containing four domains bound Ca2+ in the manner expected from the number and nature of EF hands. In contrast, aM bound only two Ca2+, suggesting that deletion of domain I may have disrupted binding in one of the remaining three domains, and did not activate the three enzymes. The kinetics of activation of PDE by CaM, TaM, and TaM-BMI were identical. Although cTnC and CaT could maximally activate PDE, the Kact for these mutants were greater than 2000 times than for CaM. All mutated proteins except CaT were poor activators of CaM kinase II and this protein activated the kinase to 65% that of CaM, with a nearly identical Kact. CaT and TaM, were poor agonists of MLCK. Activation of Ca2(+)-binding site I in TaM (TaM-BMI), completely prevented activation of MLCK. In addition, TaM-BMI was a potent competitive inhibitor of MLCK activation by CaM (Ki = 66 nM). We conclude 1) a domain I is necessary to activate these target enzymes, and the substitution of the corresponding region of cTnC into CaM leads to differential effects; 2) an active first Ca2(+)-binding site is not essential for activation of PDE and the primary sequence of the first domain of CaM need not be highly conserved; 3) for CaM kinase II, determinants in the first domain are critical whereas more flexibility exists for the remaining three domains; 4) since TaM-BMI acts as a potent competitive inhibitor of MLCK binding of CaM to a target enzyme and activation can be dissociable events.
为了评估钙调蛋白（CaM）的结构域I在靶酶激活中的作用，构建了一系列CaM突变体，其中结构域I（49个氨基酸）被大量删除，或与心肌肌钙蛋白C（cTnC）的同源区域（58个氨基酸）进行交换。这些蛋白分别是：1）aM，结构域I被删除的突变型CaM；2）TaM，cTnC的第一个结构域，CaM的后三个结构域；3）TaM-BMI，与TaM相同，但通过诱变恢复了无功能的第一个Ca2+结合结构域；4）CaT，CaM的第一个结构域，cTnC的后三个结构域。对这些蛋白进行了Ca2+结合特性评估，并作为三种CaM靶酶的激活剂进行了评估，这三种靶酶分别是CaM依赖性磷酸二酯酶（PDE）、平滑肌肌球蛋白轻链激酶（MLCK）和CaM依赖性多功能蛋白激酶（CaM激酶II）。含有四个结构域的嵌合蛋白以EF手的数量和性质所预期的方式结合Ca2+。相比之下，aM仅结合两个Ca2+，这表明结构域I的缺失可能破坏了其余三个结构域之一的结合，并且不能激活这三种酶。CaM、TaM和TaM-BMI对PDE的激活动力学是相同的。虽然cTnC和CaT可以最大程度地激活PDE，但这些突变体的激活常数（Kact）比CaM大2000倍以上。除CaT外，所有突变蛋白都是CaM激酶II的弱激活剂，而CaT能将该激酶激活至CaM激活水平的65%，且Kact几乎相同。CaT和TaM是MLCK的弱激动剂。TaM（TaM-BMI）中Ca2+结合位点I的激活完全阻止了MLCK的激活。此外，TaM-BMI是CaM激活MLCK的有效竞争性抑制剂（抑制常数Ki = 66 nM）。我们得出以下结论：1）结构域I是激活这些靶酶所必需的，将cTnC的相应区域替换到CaM中会产生不同的效应；2）一个活性的第一个Ca2+结合位点对于PDE的激活不是必需的，CaM第一个结构域的一级序列不需要高度保守；3）对于CaM激酶II，第一个结构域中的决定因素至关重要，而其余三个结构域则具有更大的灵活性；4）由于TaM-BMI作为CaM与靶酶MLCK结合的有效竞争性抑制剂，激活可能是可分离的事件。
