Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts.
Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M- to Z-lines. We have cloned and sequenced the C-terminal region of the titin molecule, which is an integral part of M-lines and forms intimate contacts with the 165 and 190 kDa M-line proteins. In contrast to the regular motif patterns of the A-band portion of titin, the 5.7 kb of titin sequences from the M-line show a complex structure of immunoglobulin-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi-phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP-specific kinases is regulated during differentiation, and that this may control the assembly of M-line proteins into regular structures during myogenesis.
Gautel
M
M
European Molecular Biology Laboratory, Biological Structures Division, Heidelberg.
Leonard
K
K
Labeit
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D064211:Connectin; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism*; D009132:Muscles / Q000166:cytology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases