Calcium/calmodulin-dependent protein kinase kinase: identification of regulatory domains.
Calcium/calmodulin-dependent protein kinase kinase: identification of regulatory domains.
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钙/钙调蛋白依赖性蛋白激酶激酶：调节域的鉴定。
DOI：
10.1021/bi971348i
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Soderling,TR
中科院分区：
文献类型：
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作者：
Tokumitsu,H;Wayman,GA;Muramatsu,M;Soderling,TR
我们最近克隆了钙调蛋白依赖性蛋白激酶激酶（CaM-KK），其磷酸化并激活CaM-KI和CaM-KIV [Tokumitsu，H.，Enslen，H.，和Soderling，T. R.（1995）J.Biol.Chem.270，19320 - 19324]。在本研究中，我们已经确定了其监管钙调素结合和自身抑制域（CBD和AID，分别）使用一系列的COOH-末端截短和定点突变体在COS-7细胞中表达。截短突变体CaM-KK1 - 463与野生型酶（CaM-KK1 - 505）类似地激活CaM-KIV并结合CaM; CaM-KK1 - 448不结合CaM且基本上无活性; CaM-KK1 - 434也不结合CaM，但在Ca2 +/CaM不存在的情况下激活CaM-KIV的CaM非依赖性突变体。在455 RKR、448 ILV或443 SWT位置替换三重负电荷（Asp）可阻断CaM结合，并抑制70 - 90%的CaM-KK活性。DDD突变体438VKL和435KNS具有部分Ca~（2+）/Ca~（2+）非依赖性活性。这些结果鉴定了CaM-KK中残基430和460之间的重叠AID和CBD，类似于其他CaM-Ks。与这一分配一致，对应于残基438 − 463的合成肽以Ca2+依赖性方式结合CaM，aK在低纳摩尔范围内。此外，cAMP-激酶对CaM-KK中CBD COOH-末端的Ser458的磷酸化抑制了随后的CaM结合[Wayman，G.，Tokumitsu，H.，和Soderling，T. R.（1997）J.Biol.Chem.272，16073 - 16076]，被Ca2 +/CaM与CaM-KK的预先结合所阻断。
We recently cloned a calmodulin-dependent protein kinase kinase (CaM-KK) which phosphorylates and activates CaM-KI and CaM-KIV [Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995)J. Biol. Chem.270, 19320−19324]. In the present study, we have identified its regulatory CaM-binding and autoinhibitory domains (CBD and AID, respectively) using a series of COOH-terminal truncations and site-directed mutants expressed in COS-7 cells. Truncation mutant CaM-KK1-463activated CaM-KIV and bound CaM similar to wild-type enzyme (CaM-KK1-505); CaM-KK1-448did not bind CaM and was largely inactive; and CaM-KK1-434also did not bind CaM but activated a CaM-independent mutant of CaM-KIV in the absence of Ca2+/CaM. Substitution of triple negative charges (Asp) at positions455RKR,448ILV, or443SWT blocked CaM binding and suppressed by 70−90% CaM-KK activities. Mutants438VKL and435KNS to DDD exhibited partial Ca2+/CaM-independent activities. These results identify overlapping AID and CBD between residues 430 and 460 in CaM-KK, similar to other CaM-Ks. Consistent with this assignment, the synthetic peptide corresponding to residues 438−463 bound CaM in a Ca2+-dependent manner with aKdin the low nanomolar range. Furthermore, phosphorylation by cAMP-kinase of Ser458at the COOH-terminus of the CBD in CaM-KK, which suppresses subsequent CaM binding [Wayman, G., Tokumitsu, H., and Soderling, T. R. (1997)J. Biol. Chem. 272, 16073−16076], was blocked by prior binding of Ca2+/CaM to CaM-KK.