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

PakB is a p21-activated kinase that phosphorylates and activates class I myosins in the social amoeba Dictyostelium discoideum. PakB co-localizes with myosin-I to actin-rich regions of the cell, including macropinocytic cups and the leading edge of migrating cells. Here we show that the cellular localization of PakB depends on the N-terminal region which contains an actin filament binding module and two proline-rich motifs that interact with the SH3 domain of actin-binding protein 1 (dAbp1). dAbp1 co-localized with PakB to actin-rich sites, but in PakBˉ (PakB null) cells dAbp1 adopted a diffuse cytosolic distribution. Overexpression of dAbp1 in PakBˉ cells produced SH3 domain-dependent defects in early development, cell polarization and chemotaxis. We conclude that PakB plays a critical role in regulating the cellular functions of the dAbp1 SH3 domain. PakBˉ cells exhibited a disrupted cortical actin layer and were extremely sensitive to external stresses induced by compression and electroporation. PakBˉ cells showed severe chemotaxis defects when forced to migrate under agarose. The defects were rescued by expression of full-length PakB but not an N-terminal fragment of PakB. The results suggest that loss of PakB kinase activity is responsible for the cortical defects. Immunoblot analysis showed that phosphorylation of MyoD at the TEDS site was significantly reduced in PakBˉ cells. We propose that activation of myosin-I motor activity by PakB plays a critical role in stabilizing the cortical actin cytoskeleton. D. discoideum myosin-II heavy chain kinase A (MHCK-A) is a member of the alpha-kinase family. Competition experiments with Mant-ADP showed that MHCK-A bound ATP with Ki values of 18 and 160 µM in the presence and absence of Mg2+, respectively. ADP and AMP bound 3-fold and 9-fold more weakly than ATP, respectively. The results show that Mg2+ and the nucleotide phosphoryl groups substantially contribute to binding. Mutations of residues in the Pi-pocket and N/D-loop reduced the binding affinity for MgATP, showing that both regulatory sites are coupled to the active site. Phosphorylation of SPOT peptide arrays with MHCK-A revealed a consensus sequence of T-φ/K-φ/K-K/R and showed also phosphorylation of non-Thr-containing peptides. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-08-21 14:02:30.015