Studies on Dictyostelium Discoideum Myosin-I and Myosin-II Heavy Chain Kinases

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Date
2013-08-21
Authors
Yang, Yidai
Keyword
MHCK-A , Dictyostelium discoideum , PakB , dAbp1
Abstract
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.
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