Caldesmon Binding to Actin Is Regulated by Calmodulin and Phosphorylation via Different Mechanisms

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• 作者：Renjian Huang ; Liansheng Li ; Hongqiu Guo ; and C.-L. Albert Wang
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：March 11, 2003
• 年：2003
• 卷：42
• 期：9
• 页码：2513 - 2523
• 全文大小：283K
• 年卷期：v.42,no.9(March 11, 2003)
• ISSN：1520-4995

文摘

Smooth muscle caldesmon (CaD) binds F-actin and inhibits actomyosin ATPase activity. Theinhibition is reversed by Ca²⁺/calmodulin (CaM). CaD is also phosphorylated upon stimulation at sitesspecific for mitogen-activated protein kinases (MAPKs). Because of these properties, CaD is thought tobe involved in the regulation of smooth muscle contraction. The molecular mechanism of the reversal ofinhibition is not well understood. We have expressed His₆-tagged fragments containing the sequence ofthe C-terminal region of human (from M563 to V793) and chicken (from M563 to P771) CaD as well asa variant of the chicken isoform with a Q766C point mutation. By cleavages with proteases, followed byhigh-speed cosedimentation with F-actin and mass spectrometry, we found that within the C-terminalregion of CaD there are multiple actin contact points forming two clusters. Intramolecular fluorescenceresonance energy transfer between probes attached to cysteine residues (the endogenous C595 and theengineered C766) located in these two clusters revealed that the C-terminal region of CaD is elongated,but it becomes more compact when bound to actin. Binding of CaM restores the elongated conformationand facilitates dissociation of the C-terminal CaD fragment from F-actin. When the CaD fragment wasphosphorylated with a MAPK, only one of the two actin-binding clusters dissociated from F-actin, whereasthe other remained bound. Taken together, these results demonstrate that while both Ca²⁺/CaM and MAPKphosphorylation govern CaD's function via a conformational change, the regulatory mechanisms aredifferent.