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

This work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis. / text