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Cellular distribution and functions of wild-type and constitutively activated Dictyostelium PakB

Marc de la Roche1, Amjad Mahasneh, Sheu-Fen Lee

  • 1Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.

Molecular Biology of the Cell
|October 29, 2004
PubMed

Insights

Dictyostelium PakB, a p21-activated kinase, interacts with Rac proteins and localizes to cellular structures involved in cell motility and division. Activated PakB affects cell division and enhances phagocytosis and pinocytosis.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Dictyostelium PakB, a myosin I heavy chain kinase, is identified as a member of the p21-activated kinase (PAK) family.
  • PakB's interaction with various Dictyostelium Rac proteins suggests a role in regulating actin dynamics.

Purpose of the Study:

  • To investigate the cellular localization and function of Dictyostelium PakB.
  • To understand the role of PakB in cell migration, phagocytosis, pinocytosis, and cytokinesis.

Main Methods:

  • Two-hybrid assays were employed to identify PakB interacting proteins, specifically Dictyostelium Rac proteins.
  • Localization studies using fluorescently tagged PakB variants (wild-type, N-terminally fused to GFP, C-terminally truncated, and activated forms) were performed in Dictyostelium cells.
  • Phenotypic analysis of PakB-null cells and cells expressing activated PakB was conducted.

Main Results:

  • PakB interacts with multiple Dictyostelium Rac proteins, including Rac1a/b/c, RacA, RacB, RacC, and RacF1.
  • Wild-type PakB shows cytosolic distribution with enrichment at the leading edge and phagocytic/macropinocytic cups.
  • Activated PakB strongly localizes to the cell cortex, macropinocytic cups, posterior of migrating cells, and cleavage furrow, indicating association with actin filaments.
  • PakB-null cells exhibit no major defects, but activated PakB expression impairs cytokinesis and increases phagocytosis and pinocytosis rates.

Conclusions:

  • The N-terminus of activated PakB tightly associates with cortical actin filaments.
  • PakB plays a role in regulating cell division, phagocytosis, and pinocytosis in Dictyostelium.
  • PakB's localization and functional effects highlight its importance in cytoskeletal regulation and cell dynamics.

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