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1.
Blood ; 114(19): 4209-20, 2009 Nov 05.
Article in English | MEDLINE | ID: mdl-19638624

ABSTRACT

The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several beta(2) integrin (CD11/CD18)-mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein-tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during beta(2) integrin-mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised beta(2) integrin-mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor alpha. Functional analysis showed that the down-regulation of mAbp1 diminished the number of beta(2) integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.


Subject(s)
CD18 Antigens/physiology , Microfilament Proteins/physiology , Neutrophils/physiology , src Homology Domains/physiology , Animals , Cell Adhesion/physiology , Escherichia coli/pathogenicity , HL-60 Cells , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Microfilament Proteins/antagonists & inhibitors , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Phagocytosis/physiology , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , RNA Interference , Receptors, IgG/metabolism , Salmonella typhimurium/pathogenicity , Syk Kinase , src Homology Domains/genetics
2.
Mol Microbiol ; 68(6): 1438-49, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18410496

ABSTRACT

The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N-glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N-glycan to be directly involved in Mid2p sensing.


Subject(s)
Calcium-Binding Proteins/metabolism , Cell Wall/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Cell Wall/chemistry , Cell Wall/genetics , Glycosylation , Intracellular Signaling Peptides and Proteins , Membrane Glycoproteins , Membrane Proteins/chemistry , Membrane Proteins/genetics , Phenotype , Protein Structure, Tertiary , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Deletion
3.
PLoS One ; 2(11): e1132, 2007 Nov 07.
Article in English | MEDLINE | ID: mdl-17987119

ABSTRACT

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in beta(2) integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during beta2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class I(A). Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110delta of PI3K class I(A) as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110delta to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of beta2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110delta signaling for beta2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.


Subject(s)
Chemotaxis, Leukocyte , Intracellular Signaling Peptides and Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Pseudopodia , Base Sequence , DNA Primers , Electroporation , HL-60 Cells , Humans , Microscopy, Confocal , Syk Kinase
4.
Blood ; 108(12): 3919-27, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-16882714

ABSTRACT

Leukocyte adhesion via beta(2) integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe-induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk(-/-) neutrophils from wild-type mice reconstituted with Syk(-/-) bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk(-/-) bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk(-/-) neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for beta(2) integrin-mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.


Subject(s)
CD11 Antigens/metabolism , CD18 Antigens/metabolism , Cell Movement , Intracellular Signaling Peptides and Proteins/metabolism , Neutrophils/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-vav/metabolism , Animals , Arthus Reaction/genetics , Arthus Reaction/metabolism , Binding Sites , CD11 Antigens/genetics , CD18 Antigens/genetics , Cell Movement/drug effects , Cell Movement/genetics , HL-60 Cells , Humans , In Vitro Techniques , Inflammation/genetics , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/deficiency , Mice , Mice, Knockout , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neoplasm Proteins , Neutrophil Infiltration/genetics , Neutrophils/cytology , Protein-Tyrosine Kinases/deficiency , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-vav/genetics , Pseudopodia/genetics , Pseudopodia/metabolism , Syk Kinase , cdc42 GTP-Binding Protein/metabolism
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