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A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Chang FS, Han GS, Carman GM, Blumer KJ - J. Cell Biol. (2005)

Bottom Line: Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility.Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis.Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

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Catalytically inactive Lsb6 point mutants. (A) Schematic of Lsb6 indicating the two halves of the kinase domain (shaded). Residues changed singly or together to alanine to generate kinase-inactive forms of Lsb6 are indicated. (B) Expression of HA-tagged wild-type or mutant Lsb6 in lsb6Δ cells detected with an anti-HA antibody. (C) PI 4-kinase activity detected in extracts of lsb6Δ cells carrying wild-type or mutant Lsb6; data shown are the average of two experiments.
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fig3: Catalytically inactive Lsb6 point mutants. (A) Schematic of Lsb6 indicating the two halves of the kinase domain (shaded). Residues changed singly or together to alanine to generate kinase-inactive forms of Lsb6 are indicated. (B) Expression of HA-tagged wild-type or mutant Lsb6 in lsb6Δ cells detected with an anti-HA antibody. (C) PI 4-kinase activity detected in extracts of lsb6Δ cells carrying wild-type or mutant Lsb6; data shown are the average of two experiments.

Mentions: To determine whether Lsb6 synthesizes PI4P to promote endosome motility, we generated and analyzed several point mutants defective in PI 4-kinase activity. Like other type II PI 4-kinases, the kinase domain of Lsb6 is interrupted by a linker such that motifs 1, 2, 3, and 4 of the catalytic domain are located in the NH2-terminal portion of the molecule and motifs 6 and 7 in the COOH-terminal region (Fig. 3 A and Fig. S3). Flanking the two kinase subdomains are noncatalytic NH2- and COOH-terminal extensions. Studies of the rat type II PI 4-kinase have identified residues required specifically for catalysis; substitution of any of these residues eliminates enzyme activity (Barylko et al., 2002). Accordingly, we targeted the equivalent amino acids either singly or together in NH2-terminally HA-tagged Lsb6 to yield the following mutants: K192M, D387A, N392A, D413A, and a quadruple mutant (4KD) bearing all four of these substitutions (Fig. 3 A). Each mutant form of HA-Lsb6 of the expected molecular mass was well expressed (Fig. 3 B). We used assay conditions optimized to detect the PI 4-kinase activity of Lsb6 and to minimize activity of the other PI 4-kinases Stt4 and Pik1 (Han et al., 2002). Under these conditions, we found that extracts from cells expressing wild-type HA-Lsb6 showed robust PI 4-kinase activity relative to the low level of activity detected in extracts from lsb6Δ cells expressing vector alone (Fig. 3 C). In contrast, extracts derived from cells expressing any of the kinase domain point mutants lacked PI 4-kinase activity above the background (Fig. 3 C).


A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

Chang FS, Han GS, Carman GM, Blumer KJ - J. Cell Biol. (2005)

Catalytically inactive Lsb6 point mutants. (A) Schematic of Lsb6 indicating the two halves of the kinase domain (shaded). Residues changed singly or together to alanine to generate kinase-inactive forms of Lsb6 are indicated. (B) Expression of HA-tagged wild-type or mutant Lsb6 in lsb6Δ cells detected with an anti-HA antibody. (C) PI 4-kinase activity detected in extracts of lsb6Δ cells carrying wild-type or mutant Lsb6; data shown are the average of two experiments.
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Related In: Results  -  Collection

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fig3: Catalytically inactive Lsb6 point mutants. (A) Schematic of Lsb6 indicating the two halves of the kinase domain (shaded). Residues changed singly or together to alanine to generate kinase-inactive forms of Lsb6 are indicated. (B) Expression of HA-tagged wild-type or mutant Lsb6 in lsb6Δ cells detected with an anti-HA antibody. (C) PI 4-kinase activity detected in extracts of lsb6Δ cells carrying wild-type or mutant Lsb6; data shown are the average of two experiments.
Mentions: To determine whether Lsb6 synthesizes PI4P to promote endosome motility, we generated and analyzed several point mutants defective in PI 4-kinase activity. Like other type II PI 4-kinases, the kinase domain of Lsb6 is interrupted by a linker such that motifs 1, 2, 3, and 4 of the catalytic domain are located in the NH2-terminal portion of the molecule and motifs 6 and 7 in the COOH-terminal region (Fig. 3 A and Fig. S3). Flanking the two kinase subdomains are noncatalytic NH2- and COOH-terminal extensions. Studies of the rat type II PI 4-kinase have identified residues required specifically for catalysis; substitution of any of these residues eliminates enzyme activity (Barylko et al., 2002). Accordingly, we targeted the equivalent amino acids either singly or together in NH2-terminally HA-tagged Lsb6 to yield the following mutants: K192M, D387A, N392A, D413A, and a quadruple mutant (4KD) bearing all four of these substitutions (Fig. 3 A). Each mutant form of HA-Lsb6 of the expected molecular mass was well expressed (Fig. 3 B). We used assay conditions optimized to detect the PI 4-kinase activity of Lsb6 and to minimize activity of the other PI 4-kinases Stt4 and Pik1 (Han et al., 2002). Under these conditions, we found that extracts from cells expressing wild-type HA-Lsb6 showed robust PI 4-kinase activity relative to the low level of activity detected in extracts from lsb6Δ cells expressing vector alone (Fig. 3 C). In contrast, extracts derived from cells expressing any of the kinase domain point mutants lacked PI 4-kinase activity above the background (Fig. 3 C).

Bottom Line: Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility.Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis.Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

Show MeSH
Related in: MedlinePlus