CHARACTERIZATION OF A 5.4 KB CDNA FRAGMENT FROM THE Z-LINE REGION OF RABBIT CARDIAC TITIN REVEALS PHOSPHORYLATION SITES FOR PROLINE-DIRECTED KINASES

Titin is an approximately 3 MDa protein that spans from the M- to the Z-line in the sarcomeres of vertebrate striated muscle. The protein is presumably encoded by unusually large mRNAs of 70-80 kb, Although tit in has been studied by several laboratories, barely more than half of the cDNA sequence (approximate to 45 kb) has been published, most of i t obtained from the A-band and M-line region (corresponding to the C-t erminal half of the molecule), A special cDNA library was constructed using size selected total RNA from adult rabbit cardiac muscle in orde r to obtain sequence data from titin's unknown N-terminal region, A mo noclonal antibody (T12), which binds to an epitope close to the Z-line , was used to identify initial cDNA clones, Additional overlapping clo nes were isolated and sequenced yielding a 5.4 kb contig, The encoded polypeptide contains 16 Type-II domains and four unique intervening se gments, Polyclonal sera, raised against an expressed protein fragment encoded by the 5' end of the contig, strongly stained the Z-line of my ofibrils of different species. However, the sequence of this fragment is 83% identical at the amino acid level with the previously reported C-terminal (i,e, M-line) end of chicken embryonic skeletal muscle titi n, The expressed protein fragment could be phosphorylated in vitro by embryonic skeletal muscle extract and by the purified proline-directed kinase ERK1, presumably at the xSPxR recognition sites located in the first interdomain segment.