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

No description available.

Clathrin-coated pits on the plasma membrane invaginate into coated vesicles to internalize receptors and membrane. The clathrin adaptor epsin contains an aminoterminal ENTH domain that binds PI(4,5)P₂ and a carboxy-terminal domain that binds clathrin, and accessory proteins such as AP2. Here, we assessed how inter- and intramolecular factors affect the contribution of epsin to coated-pit function in living cells. We found Dictyostelium epsin was not required for global clathrin function, but plays an essential role in spore development. We demonstrated that clathrin, but not AP2, was critical for epsin to associate with clathrin-coated pits. We found that the carboxy-terminal region of epsin was essential, but not sufficient, for targeting epsin within clathrin-coated pits on the plasma membrane. In addition to targeting epsin to the membrane, the amino-terminal ENTH domain regulates the interaction between epsin and clathrin, an essential property that cannot be replaced by an alternate PI(4,5)P₂ binding domain. Moreover, the ENTH domain facilitates the functional interaction between clathrin and actin during late stages of endocytosis, possibly by regulating the activity of the adaptor Hip1r. Both the ability to bind PI(4,5)P₂ and another function mediated by residue T107 are critical for the activity of the ENTH domain. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits. Furthermore, we propose that the ENTH domain of epsin facilitates the membrane recruitment and phosphorylation of Hip1r, which in turn mediates the productive interaction of clathrin with the actin cytoskeleton at the plasma membrane. / text

The function of CABP1, a novel cAMP-binding protein in Dictyostelium discoideum, has been investigated. Previously, it has been shown that the molecular weight of CABP1 varies in different strains and that this variation is correlated with differences in response to cAMP. Sequence analysis showed that compared to its counterpart in strain AX2, the gene encoding CABP1 of strain V12M2 is missing 27 bp. AX2 transformants expressing the capA gene of V12M2 exhibited no change in phenotype, suggesting that CABP1 is not involved in the features examined or that the V12M2 variant is recessive. Disruption of capA by homologous recombination was attempted with no success, suggesting that this gene might be essential. Proteins interacting with CABP1 have been identified by immunoprecipitation using specific antibodies, CABP1 was also found to copurify with annexin VII, a calcium-dependent phospholipid-binding protein, suggesting that CABP1 and annexin VII might interact.

Universiẗat, Diss., 2006--Frankfurt (Main).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Universiẗat, Diss., 2001--München.

Thesis (Ph. D.)--University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

University, Diss., 2004--Köln.