Different head environments in tarantula thick filaments support a cooperative activation process
Department of Cell and Developmental Biology
Actins; Amino Acid Sequence; Animals; *Arachnida; Arthropod Proteins; Glycerol; Models, Molecular; Molecular Sequence Data; Myosin-Light-Chain Kinase; Myosins; Phosphorylation; Protein Kinase C; Serine; Urea
Biophysics | Cell Biology | Cellular and Molecular Physiology
Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). Structural analysis of relaxed tarantula thick filaments shows that the RLCs of the interacting free and blocked myosin heads are in different environments. This and other data suggested a phosphorylation mechanism in which Ser-35 of the free head is exposed and constitutively phosphorylated by protein kinase C, whereas the blocked head is hidden and unphosphorylated; on activation, myosin light chain kinase phosphorylates the monophosphorylated free head followed by the unphosphorylated blocked head, both at Ser-45. Our goal was to test this model of phosphorylation. Mass spectrometry of quickly frozen, intact muscles showed that only Ser-35 was phosphorylated in the relaxed state. The location of this constitutively phosphorylated Ser-35 was analyzed by immunofluorescence, using antibodies specific for unphosphorylated or phosphorylated Ser-35. In the relaxed state, myofibrils were labeled by anti-pSer-35 but not by anti-Ser-35, whereas in rigor, labeling was similar with both. This suggests that only pSer-35 is exposed in the relaxed state, while in rigor, Ser-35 is also exposed. In the interacting-head motif of relaxed filaments, only the free head RLCs are exposed, suggesting that the constitutive pSer-35 is on the free heads, consistent with the proposed mechanism.
DOI of Published Version
Sulbarán G, Biasutto A, Alamo L, Riggs C, Pinto A, Méndez F, Craig R, Padrón R. Different head environments in tarantula thick filaments support a cooperative activation process. Biophys J. 2013 Nov 5;105(9):2114-22. doi:10.1016/j.bpj.2013.09.001. Link to article on publisher's site
Sulbaran, Guidenn; Biasutto, Antonio; Alamo, Lorenzo; Riggs, Claire; Pinto, Antonio; Mendez, Franklin; Craig, Roger; and Padron, Raul, "Different head environments in tarantula thick filaments support a cooperative activation process" (2013). Craig Lab Publications. 27.