Structure-Function Studies on the Novel Alpha-Kinase Family
Samimi Gharaei, Mojdeh
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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.