title: Different head environments in tarantula thick filaments support a cooperative activation process.
abstract: 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.
authors.affiliation: Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Venezuela; Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
authors.forename: Guidenn
authors.initials: G
authors.lastname: Sulbarán
authors.forename: Antonio
authors.initials: A
authors.lastname: Biasutto
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Alamo
authors.forename: Claire
authors.initials: C
authors.lastname: Riggs
authors.forename: Antonio
authors.initials: A
authors.lastname: Pinto
authors.forename: Franklin
authors.initials: F
authors.lastname: Méndez
authors.forename: Roger
authors.initials: R
authors.lastname: Craig
authors.forename: Raúl
authors.initials: R
authors.lastname: Padrón
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001097:Arachnida; D060829:Arthropod Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005990:Glycerol / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014508:Urea / Q000737:chemistry
chemical_list: D000199:Actins; D060829:Arthropod Proteins; D012694:Serine; D014508:Urea; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D005990:Glycerol