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Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Stupack DG, Li E, Silletti SA, Kehler JA, Geahlen RL, Hahn K, Nemerow GR, Cheresh DA - J. Cell Biol. (1999)

Bottom Line: Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation.Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes.In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

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Activation of Syk  and PI(3)K after integrin ligation. (A) NP-40 lysates of  LCL plated on wells coated  with either BSA (B), monomeric penton base (PB1) or  pentameric penton base (PB5)  generated 15 min after plating were subjected to immunoprecipitation for Syk,  followed by immunoblot  analysis for phosphotyrosine  or Syk with monoclonal antibodies 4G10 or 4D10, respectively. (B) Autokinase activity of immunoprecipitated  Syk was assessed by in vitro  kinase assay after immunoprecipitation. The mean relative increase and SEM from  three separate experiments is  plotted. (C) NP-40 lysates of  LCL plated on wells coated  with either BSA or monomeric or pentameric PB were  similarly subjected to immunoprecipitation for the p85  subunit of PI(3)K, followed  by immunoblotting analysis for phosphotyrosine with monoclonal antibody 4G10. (D) The p85-associated PI(3)K activity  was assessed after immunoprecipitation of p85 and the addition  of exogenous 4,5 PIP2 and 5 μCi 32P-ATP. Lipids were resolved  on TLC-silica plates (1:1:1, CHCl3, CH3OH, 1 M HCl). The mean  relative increase and SEM from three separate experiments is  plotted.
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Figure 6: Activation of Syk and PI(3)K after integrin ligation. (A) NP-40 lysates of LCL plated on wells coated with either BSA (B), monomeric penton base (PB1) or pentameric penton base (PB5) generated 15 min after plating were subjected to immunoprecipitation for Syk, followed by immunoblot analysis for phosphotyrosine or Syk with monoclonal antibodies 4G10 or 4D10, respectively. (B) Autokinase activity of immunoprecipitated Syk was assessed by in vitro kinase assay after immunoprecipitation. The mean relative increase and SEM from three separate experiments is plotted. (C) NP-40 lysates of LCL plated on wells coated with either BSA or monomeric or pentameric PB were similarly subjected to immunoprecipitation for the p85 subunit of PI(3)K, followed by immunoblotting analysis for phosphotyrosine with monoclonal antibody 4G10. (D) The p85-associated PI(3)K activity was assessed after immunoprecipitation of p85 and the addition of exogenous 4,5 PIP2 and 5 μCi 32P-ATP. Lipids were resolved on TLC-silica plates (1:1:1, CHCl3, CH3OH, 1 M HCl). The mean relative increase and SEM from three separate experiments is plotted.

Mentions: Although LCL failed to express detectable levels of FAK (data not shown), they expressed both Syk and PI(3)K. Attachment of LCL to pentavalent PB resulted in enhancement of Syk phosphorylation compared with Syk isolated from cells plated on the monomeric substrate or on BSA (4.5 ± 1.7 fold) (Fig. 6 A). Importantly, there was a corresponding two- to threefold increase in Syk kinase activity in cells adherent to the pentamer compared with cells plated on the monomer or the nonadherent substrate protein BSA (Fig. 6 B). In contrast, an increase in phosphorylation of the p85 subunit of PI(3)K (Fig. 6 C) and PI(3)K activity (Fig. 6 D) was observed in cells interacting with either pentamer or monomer relative to cells plated on BSA. These data suggest that lymphoid cell PI(3)K becomes activated with a low threshold of integrin ligation in general, whereas integrin ligation leading to (stable) adhesion is specifically associated with activation of Syk.


Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Stupack DG, Li E, Silletti SA, Kehler JA, Geahlen RL, Hahn K, Nemerow GR, Cheresh DA - J. Cell Biol. (1999)

Activation of Syk  and PI(3)K after integrin ligation. (A) NP-40 lysates of  LCL plated on wells coated  with either BSA (B), monomeric penton base (PB1) or  pentameric penton base (PB5)  generated 15 min after plating were subjected to immunoprecipitation for Syk,  followed by immunoblot  analysis for phosphotyrosine  or Syk with monoclonal antibodies 4G10 or 4D10, respectively. (B) Autokinase activity of immunoprecipitated  Syk was assessed by in vitro  kinase assay after immunoprecipitation. The mean relative increase and SEM from  three separate experiments is  plotted. (C) NP-40 lysates of  LCL plated on wells coated  with either BSA or monomeric or pentameric PB were  similarly subjected to immunoprecipitation for the p85  subunit of PI(3)K, followed  by immunoblotting analysis for phosphotyrosine with monoclonal antibody 4G10. (D) The p85-associated PI(3)K activity  was assessed after immunoprecipitation of p85 and the addition  of exogenous 4,5 PIP2 and 5 μCi 32P-ATP. Lipids were resolved  on TLC-silica plates (1:1:1, CHCl3, CH3OH, 1 M HCl). The mean  relative increase and SEM from three separate experiments is  plotted.
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Related In: Results  -  Collection

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Figure 6: Activation of Syk and PI(3)K after integrin ligation. (A) NP-40 lysates of LCL plated on wells coated with either BSA (B), monomeric penton base (PB1) or pentameric penton base (PB5) generated 15 min after plating were subjected to immunoprecipitation for Syk, followed by immunoblot analysis for phosphotyrosine or Syk with monoclonal antibodies 4G10 or 4D10, respectively. (B) Autokinase activity of immunoprecipitated Syk was assessed by in vitro kinase assay after immunoprecipitation. The mean relative increase and SEM from three separate experiments is plotted. (C) NP-40 lysates of LCL plated on wells coated with either BSA or monomeric or pentameric PB were similarly subjected to immunoprecipitation for the p85 subunit of PI(3)K, followed by immunoblotting analysis for phosphotyrosine with monoclonal antibody 4G10. (D) The p85-associated PI(3)K activity was assessed after immunoprecipitation of p85 and the addition of exogenous 4,5 PIP2 and 5 μCi 32P-ATP. Lipids were resolved on TLC-silica plates (1:1:1, CHCl3, CH3OH, 1 M HCl). The mean relative increase and SEM from three separate experiments is plotted.
Mentions: Although LCL failed to express detectable levels of FAK (data not shown), they expressed both Syk and PI(3)K. Attachment of LCL to pentavalent PB resulted in enhancement of Syk phosphorylation compared with Syk isolated from cells plated on the monomeric substrate or on BSA (4.5 ± 1.7 fold) (Fig. 6 A). Importantly, there was a corresponding two- to threefold increase in Syk kinase activity in cells adherent to the pentamer compared with cells plated on the monomer or the nonadherent substrate protein BSA (Fig. 6 B). In contrast, an increase in phosphorylation of the p85 subunit of PI(3)K (Fig. 6 C) and PI(3)K activity (Fig. 6 D) was observed in cells interacting with either pentamer or monomer relative to cells plated on BSA. These data suggest that lymphoid cell PI(3)K becomes activated with a low threshold of integrin ligation in general, whereas integrin ligation leading to (stable) adhesion is specifically associated with activation of Syk.

Bottom Line: Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation.Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes.In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

Show MeSH
Related in: MedlinePlus