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Phosphorylated pleckstrin induces cell spreading via an integrin-dependent pathway.

Roll RL, Bauman EM, Bennett JS, Abrams CS - J. Cell Biol. (2000)

Bottom Line: Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells.This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading.Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine of the University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Pleckstrin is a 40-kD phosphoprotein containing NH(2)- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, alphaIIbbeta3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant alphaIIbbeta3 Ser753Pro, or beta3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin beta-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.

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Quantification of effect of pleckstrin and integrin mutants on CHO cell spreading. CHO cells were transiently transfected with plasmids that direct the expression of GFP or wild-type pleckstrin alone (A), or wild-type pleckstrin along with either αIIb & β3 Ser753Pro or β3 Ser753Pro (B), or wild type pleckstrin along with either Tac-αIIb, Tac-β3, or Tac-β3 Ser753Pro (C). Images were captured and cell footprint size was quantified using ImageIQ software. Pleckstrin-induced cell spreading was inhibited by co-expression of αIIbβ3 Ser753Pro, β3 Ser753Pro, or Tac-β3. In contrast, Tac-αIIb or Tac-β3 Ser753Pro had little effect. Shown is the mean ± SEM from three experiments.
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Figure 6: Quantification of effect of pleckstrin and integrin mutants on CHO cell spreading. CHO cells were transiently transfected with plasmids that direct the expression of GFP or wild-type pleckstrin alone (A), or wild-type pleckstrin along with either αIIb & β3 Ser753Pro or β3 Ser753Pro (B), or wild type pleckstrin along with either Tac-αIIb, Tac-β3, or Tac-β3 Ser753Pro (C). Images were captured and cell footprint size was quantified using ImageIQ software. Pleckstrin-induced cell spreading was inhibited by co-expression of αIIbβ3 Ser753Pro, β3 Ser753Pro, or Tac-β3. In contrast, Tac-αIIb or Tac-β3 Ser753Pro had little effect. Shown is the mean ± SEM from three experiments.

Mentions: We found that, like our results using REF52 cells plated on fibrinogen, expression of wild-type αIIbβ3 in CHO cells does not influence pleckstrin-mediated cell spreading, whereas spreading was abrogated by αIIbβ3 S752P. Moreover, β3 S752P alone abrogated the pleckstrin effect (Fig. 6A and Fig. B). Because β3 is not expressed on the cell surface unless it is coupled to an α-subunit, it is likely that β3 S752P is associated with the widely expressed αv integrin subunit (Kolodziej et al. 1991). This implies that β3 Ser753Pro inhibits pleckstrin-mediated spreading by disrupting the function of an endogenous β3-containing integrin (such as αvβ3).


Phosphorylated pleckstrin induces cell spreading via an integrin-dependent pathway.

Roll RL, Bauman EM, Bennett JS, Abrams CS - J. Cell Biol. (2000)

Quantification of effect of pleckstrin and integrin mutants on CHO cell spreading. CHO cells were transiently transfected with plasmids that direct the expression of GFP or wild-type pleckstrin alone (A), or wild-type pleckstrin along with either αIIb & β3 Ser753Pro or β3 Ser753Pro (B), or wild type pleckstrin along with either Tac-αIIb, Tac-β3, or Tac-β3 Ser753Pro (C). Images were captured and cell footprint size was quantified using ImageIQ software. Pleckstrin-induced cell spreading was inhibited by co-expression of αIIbβ3 Ser753Pro, β3 Ser753Pro, or Tac-β3. In contrast, Tac-αIIb or Tac-β3 Ser753Pro had little effect. Shown is the mean ± SEM from three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2150702&req=5

Figure 6: Quantification of effect of pleckstrin and integrin mutants on CHO cell spreading. CHO cells were transiently transfected with plasmids that direct the expression of GFP or wild-type pleckstrin alone (A), or wild-type pleckstrin along with either αIIb & β3 Ser753Pro or β3 Ser753Pro (B), or wild type pleckstrin along with either Tac-αIIb, Tac-β3, or Tac-β3 Ser753Pro (C). Images were captured and cell footprint size was quantified using ImageIQ software. Pleckstrin-induced cell spreading was inhibited by co-expression of αIIbβ3 Ser753Pro, β3 Ser753Pro, or Tac-β3. In contrast, Tac-αIIb or Tac-β3 Ser753Pro had little effect. Shown is the mean ± SEM from three experiments.
Mentions: We found that, like our results using REF52 cells plated on fibrinogen, expression of wild-type αIIbβ3 in CHO cells does not influence pleckstrin-mediated cell spreading, whereas spreading was abrogated by αIIbβ3 S752P. Moreover, β3 S752P alone abrogated the pleckstrin effect (Fig. 6A and Fig. B). Because β3 is not expressed on the cell surface unless it is coupled to an α-subunit, it is likely that β3 S752P is associated with the widely expressed αv integrin subunit (Kolodziej et al. 1991). This implies that β3 Ser753Pro inhibits pleckstrin-mediated spreading by disrupting the function of an endogenous β3-containing integrin (such as αvβ3).

Bottom Line: Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells.This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading.Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine of the University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Pleckstrin is a 40-kD phosphoprotein containing NH(2)- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, alphaIIbbeta3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant alphaIIbbeta3 Ser753Pro, or beta3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin beta-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.

Show MeSH
Related in: MedlinePlus