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) almost-eq ual-to 3000 kDa) and single molecules span sarcomeres from the M- to Z -lines. We have cloned and sequenced the C-terminal region of the titi n molecule, which is an integral part of M-lines and forms intimate co ntacts with the 165 and 190 kDa M-line proteins. In contrast to the re gular motif patterns of the A-band portion of titin, the 5.7 kb of tit in sequences from the M-line show,a complex structure of immunoglobuli n-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repea ts analogous to the multi-phosphorylation repeats of neurofilament sub units H and M. In vitro phosphorylation assays with expressed titin KS P sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activi ty can be depleted from myocyte extracts by antibodies against cdc2 ki nase and p13suc1 beads, the titin KSP kinase is structurally related t o cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP- specific kinases is regulated during differentiation, and that this ma y control the assembly of M-line proteins into regular structures duri ng myogenesis.