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 titin has been studied by several laboratories, barely more than half of the cDNA sequence (approximately 45 kb) has been published, most of it obtained from the A-band and M-line region (corresponding to the C-terminal half of the molecule). A special cDNA library was constructed using size selected total RNA from adult rabbit cardiac muscle in order to obtain sequence data from titin's unknown N-terminal region. A monoclonal antibody (T12), which binds to an epitope close to the Z-line, was used to identify initial cDNA clones. Additional overlapping clones were isolated and sequenced yielding a 5.4 kb contig. The encoded polypeptide contains 16 Type-II domains and four unique intervening segments. Polyclonal sera, raised against an expressed protein fragment encoded by the 5' end of the contig, strongly stained the Z-line of myofibrils 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 titin. 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.
Department of Pediatrics, Waisman Center, University of Wisconsin-Madison 53705, USA.
M G
MG
Sebestyén
J A
JA
Wolff
M L
ML
Greaser
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D064211:Connectin; D018076:DNA, Complementary / Q000235:genetics*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D009206:Myocardium / Q000737:chemistry*; D010446:Peptide Fragments / Q000235:genetics*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid
D064211:Connectin; D018076:DNA, Complementary; D009124:Muscle Proteins; D010446:Peptide Fragments; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases