Using C. elegans to study titin and obscurin (UNC-89)

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DESCRIPTION (provided by applicant): Myofibrils contain giant polypeptides (700,000 Da--4 MDa) that have either one or two protein kinase domains. We propose to gain insights into two of these giants, titin and obscurin, by studying their C. elegans homologs, exploiting the molecular genetics available in this organism. Although the kinase domains of the giant proteins show greatest homology to myosin light chain kinases (MLCKs), the true substrates for these giant kinases are not clearly known. We recently discovered that a single gene in the worm encodes 3 titin-related polypeptides of 2.2, 1.2 and 0.3 MDa. The Ce titin protein kinase domain has protein kinase activity and is regulated by a novel mechanism. Obscurin is closely related to a C. elegans protein, UNC-89, that we described previously. Our recent data shows that some isoforms of UNC-89 have 2 MLCK-like protein kinase domains, similar to what has recently been reported for some isoforms of obscurin. In addition to signaling via protein kinase domains, obscurin and UNC-89 are likely to signal via Rho-like GTPases, since each protein has a dbl (DH) homology domain. Single molecules of the largest isoforms of mammalian titin span half a sarcomere, from M-line to Z-disc. In the I-band, various types of experiments have shown that titin is elastic. In the I-band, the "small" approximately 700 kDa "novex-3" titin interacts with obscurin. It has been suggested that a novex-3 titin / obscurin complex in the I-band is elastic, and might detect strain imposed on the sarcomere during stretch and in response, signals through the DH domain to GTPases. Our preliminary data indicates that loss-of-function for Ce titins results in paralysis and developmental arrest. We plan to characterize mutations in Ce titin, determine Ce titin's kinase substrate and other binding partners, and study structural and mechanical properties of its likely elastic elements. For UNC-89, we will explore the structure and activity of its kinase domains, identify the substrates or binding partners for its kinase domains, and identify and study binding partners for its SH3, DH and PH domains.