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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

STRUCTURE-FUNCTION STUDIES ON THE NOVEL ALPHA-KINASE FAMILY

Samimi Gharaei, MOJDEH 18 February 2010 (has links)
Dictyostelium myosin heavy chain kinase A (MHCK A) and mammalian transient receptor potential melastatin-related 7 (TRPM7) are two divergent members of a family of atypical protein kinases called the alpha kinases. The crystal structures of the alpha-kinase domains of MHCK A (A-CAT, residues 552-841) and mouse TRPM7 (TRPM7-CAT, residues 1548-1862) are very similar. In both cases a C-terminal tail (C-tail) sequence (A-CAT, residues 806-841 and TRPM7-CAT, residues 1819-1862) is missing from the crystal structure. Here I show that the unstructured C-tail is required for the catalytic activity of A-CAT and TRPM7-CAT. Truncation of the C-tail of A-CAT to residue 823 decreased kinase activity by ~98% and ATPase activity by ~97%. Truncation of the C-tail of TRPM7-CAT to residue 1827 decreased kinase activity by ~97% and ATPase activity by ~58%. Ligation of the C-tail sequence of MHCK B (residues 326-354) to A-CAT-802 (residues 552-802), fully rescued kinase activity. Alignment of the C-tail sequences of MHCK A-D revealed a conserved Gly-Thr-hydrophobic motif. Previous work has shown that in A-CAT, the conserved threonine (T825) is a site of autophosphorylation. Mutation of the T825 to alanine reduced A-CAT kinase and ATPase activities by 97%, whereas mutation to serine decreased kinase and ATPase rates by 85% and 60%, respectively. This result is consistent with the finding that A-CAT strongly prefers to phosphorylate threonine residues. Surprisingly, mutation of T825 to glutamic acid reduced kinase activity by ~93% and ATPase activity by ~96%. This result suggests that glutamic acid does not properly mimic phosphothreonine in this situation, or that the free hydroxyl group of T825 is required for the catalytic activity of A-CAT. Mutation of T825 to alanine or glutamic acid in full-length MHCK A reduced kinase activity by ~90% and ATPase activity by ~40%. Further studies are required to determine if the C-tail of TRPM7-CAT also contains an essential threonine residue. / Thesis (Master, Biochemistry) -- Queen's University, 2010-02-11 11:23:04.195

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