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Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

Mostafavi-Pour Z, Askari JA, Parkinson SJ, Parker PJ, Ng TT, Humphries MJ - J. Cell Biol. (2003)

Bottom Line: After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties.First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not.Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

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PKCα activation in cells adhering via α5β1 or α4β1. (A) Western blot analysis of total PKCα levels (top; detected with MC5 antibody) or activated PKCα (bottom; detected with PPA182 antibody) in A375-SM cells seeded on 50K+H/0 (lane 1), 50K (lane 2), H/120 (lane 3), and H/120-GAG-ABC (lane 4). (B) Densitometric analysis of A. Results are the mean ± SE of three separate experiments, of which A is representative.
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fig5: PKCα activation in cells adhering via α5β1 or α4β1. (A) Western blot analysis of total PKCα levels (top; detected with MC5 antibody) or activated PKCα (bottom; detected with PPA182 antibody) in A375-SM cells seeded on 50K+H/0 (lane 1), 50K (lane 2), H/120 (lane 3), and H/120-GAG-ABC (lane 4). (B) Densitometric analysis of A. Results are the mean ± SE of three separate experiments, of which A is representative.

Mentions: To test the involvement of PKCα in focal adhesion formation, the activation of the enzyme was measured using Western blotting. A375-SM cells adherent to either H/120, H/120-GAG-ABC, 50K, or 50K + soluble H/0 were detergent-extracted, and lysates were blotted with either a pan-anti-PKCα antibody (MC5) or an antiserum that detects activated PKCα (PPA182). Levels of PKCα were quantitated by densitometry. The level of expression of PKCα was the same for all adhesive substrata, but levels of phosphorylation differed (Fig. 5) . Thus, addition of H/0 to cells prespread on 50K potentiated the level of phosphorylation of PKCα eightfold. However, H/120 and H/120-GAG-ABC induced similar levels of PKCα phosphorylation that were indistinguishable from the basal level observed on 50K alone (Fig. 5).


Integrin-specific signaling pathways controlling focal adhesion formation and cell migration.

Mostafavi-Pour Z, Askari JA, Parkinson SJ, Parker PJ, Ng TT, Humphries MJ - J. Cell Biol. (2003)

PKCα activation in cells adhering via α5β1 or α4β1. (A) Western blot analysis of total PKCα levels (top; detected with MC5 antibody) or activated PKCα (bottom; detected with PPA182 antibody) in A375-SM cells seeded on 50K+H/0 (lane 1), 50K (lane 2), H/120 (lane 3), and H/120-GAG-ABC (lane 4). (B) Densitometric analysis of A. Results are the mean ± SE of three separate experiments, of which A is representative.
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Related In: Results  -  Collection

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fig5: PKCα activation in cells adhering via α5β1 or α4β1. (A) Western blot analysis of total PKCα levels (top; detected with MC5 antibody) or activated PKCα (bottom; detected with PPA182 antibody) in A375-SM cells seeded on 50K+H/0 (lane 1), 50K (lane 2), H/120 (lane 3), and H/120-GAG-ABC (lane 4). (B) Densitometric analysis of A. Results are the mean ± SE of three separate experiments, of which A is representative.
Mentions: To test the involvement of PKCα in focal adhesion formation, the activation of the enzyme was measured using Western blotting. A375-SM cells adherent to either H/120, H/120-GAG-ABC, 50K, or 50K + soluble H/0 were detergent-extracted, and lysates were blotted with either a pan-anti-PKCα antibody (MC5) or an antiserum that detects activated PKCα (PPA182). Levels of PKCα were quantitated by densitometry. The level of expression of PKCα was the same for all adhesive substrata, but levels of phosphorylation differed (Fig. 5) . Thus, addition of H/0 to cells prespread on 50K potentiated the level of phosphorylation of PKCα eightfold. However, H/120 and H/120-GAG-ABC induced similar levels of PKCα phosphorylation that were indistinguishable from the basal level observed on 50K alone (Fig. 5).

Bottom Line: After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties.First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not.Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The fibronectin (FN)-binding integrins alpha4beta1 and alpha5beta1 confer different cell adhesive properties, particularly with respect to focal adhesion formation and migration. After analyses of alpha4+/alpha5+ A375-SM melanoma cell adhesion to fragments of FN that interact selectively with alpha4beta1 and alpha5beta1, we now report two differences in the signals transduced by each receptor that underpin their specific adhesive properties. First, alpha5beta1 and alpha4beta1 have a differential requirement for cell surface proteoglycan engagement for focal adhesion formation and migration; alpha5beta1 requires a proteoglycan coreceptor (syndecan-4), and alpha4beta1 does not. Second, adhesion via alpha5beta1 caused an eightfold increase in protein kinase Calpha (PKCalpha) activation, but only basal PKCalpha activity was observed after adhesion via alpha4beta1. Pharmacological inhibition of PKCalpha and transient expression of dominant-negative PKCalpha, but not dominant-negative PKCdelta or PKCzeta constructs, suppressed focal adhesion formation and cell migration mediated by alpha5beta1, but had no effect on alpha4beta1. These findings demonstrate that different integrins can signal to induce focal adhesion formation and migration by different mechanisms, and they identify PKCalpha signaling as central to the functional differences between alpha4beta1 and alpha5beta1.

Show MeSH
Related in: MedlinePlus