Caldesmon binding to actin is regulated by calmodulin and phosphorylation via different mechanisms.
Smooth muscle caldesmon (CaD) binds F-actin and inhibits actomyosin ATPase activity. The inhibition is reversed by Ca2+/calmodulin (CaM). CaD is also phosphorylated upon stimulation at sites specific for mitogen-activated protein kinases (MAPKs). Because of these properties, CaD is thought to be involved in the regulation of smooth muscle contraction. The molecular mechanism of the reversal of inhibition is not well understood. We have expressed His6-tagged fragments containing the sequence of the C-terminal region of human (from M563 to V793) and chicken (from M563 to P771) CaD as well as a variant of the chicken isoform with a Q766C point mutation. By cleavages with proteases, followed by high-speed cosedimentation with F-actin and mass spectrometry, we found that within the C-terminal region of CaD there are multiple actin contact points forming two clusters. Intramolecular fluorescence resonance energy transfer between probes attached to cysteine residues (the endogenous C595 and the engineered 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 conformation and facilitates dissociation of the C-terminal CaD fragment from F-actin. When the CaD fragment was phosphorylated with a MAPK, only one of the two actin-binding clusters dissociated from F-actin, whereas the other remained bound. Taken together, these results demonstrate that while both Ca2+/CaM and MAPK phosphorylation govern CaD's function via a conformational change, the regulatory mechanisms are different.
Huang
Renjian
R
Muscle and Motility Group, Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472, USA.
Li
Liansheng
L
Guo
Hongqiu
H
Wang
C-L Albert
CL
D000199:Actins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000205:Actomyosin / Q000037:antagonists & inhibitors / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D000205:Actomyosin; D019950:Mitogen-Activated Protein Kinase 1; D000251:Adenosine Triphosphatases; D002118:Calcium