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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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Effects of wild-type or dominant-negative PKCα overexpression on integrin-mediated migration. A375-SM cells were transiently transfected with various pEGFP-PKCα constructs and then tested for their ability to migrate into wounds on either a 50K substrate in the presence of soluble H/0 or an H/120 substrate. Still images were captured at the indicated times after wounding, and separate phase, EGFP, and merged images are shown (from left to right). (A) pEGFP-PKCα-WT–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (B) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the absence of TPA; (C) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (D) pEGFP-PKCα-A25E-K368M–transfected cells on H/120 in the presence of 5 ng/ml TPA. Bars, 50 μm.
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fig8: Effects of wild-type or dominant-negative PKCα overexpression on integrin-mediated migration. A375-SM cells were transiently transfected with various pEGFP-PKCα constructs and then tested for their ability to migrate into wounds on either a 50K substrate in the presence of soluble H/0 or an H/120 substrate. Still images were captured at the indicated times after wounding, and separate phase, EGFP, and merged images are shown (from left to right). (A) pEGFP-PKCα-WT–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (B) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the absence of TPA; (C) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (D) pEGFP-PKCα-A25E-K368M–transfected cells on H/120 in the presence of 5 ng/ml TPA. Bars, 50 μm.

Mentions: Although BIM exhibits good specificity for PKC, we also attempted to perturb A375-SM migration using a dominant-negative approach. Mutant forms of PKC isoforms, tagged with GFP, were transfected into A375-SM transiently, and then their ability to close wounds was examined. Initially, wild-type pEGFP-PKCα (WT) and the double mutant pEGFP-PKCα-A25E-K368M were tested. A25E is a pseudosubstrate mutation that exposes the DAG/TPA-binding site and catalytic site. K368M is a mutation in the ATP-binding site that leaves the enzyme catalytically inactive. Thus, pEGFP-PKCα-A25E-K368M is inert, but TPA-responsive. On a 50K substrate in the presence of soluble H/0, transfection of pEGFP-PKCα-WT, either with (Fig. 8 A and Video 5) or without (unpublished data) TPA treatment 6 h after wounding, had no discernible effect on cell migration into the wound. When A375-SM cells transfected with dominant-negative pEGFP-PKCα-A25E-K368M were examined in the absence of TPA, migration again occurred normally (Fig. 8 B and Video 6). However, addition of TPA to recruit pEGFP-PKCα-A25E-K368M to the plasma membrane led to an almost complete blockade of cell movement (Fig. 8 C and Video 7). As shown most clearly in the video, GFP-expressing cells remained virtually motionless, exhibiting only occasional membrane ruffling and protrusion. However, nontransfected cells that lacked detectable GFP migrated past them and filled the wound. As an additional control, cells were transfected with pEGFP-PKCα-T497A, a substrate-binding mutant that can also act as a dominant-negative inhibitor. This mutant also prevented A375-SM cell migration on a 50K + H/0 substrate (unpublished data). To probe the contribution of PKCα to cell migration mediated by α4β1, pEGFP-PKCα-A25E-K368M–transfected cells were seeded on H/120 and wounded. In contrast to the findings on 50K + H/0, dominant-negative PKCα had no effect on wound closure (Fig. 8 D and Video 8), again suggesting that α4β1 does not depend on this enzyme.


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)

Effects of wild-type or dominant-negative PKCα overexpression on integrin-mediated migration. A375-SM cells were transiently transfected with various pEGFP-PKCα constructs and then tested for their ability to migrate into wounds on either a 50K substrate in the presence of soluble H/0 or an H/120 substrate. Still images were captured at the indicated times after wounding, and separate phase, EGFP, and merged images are shown (from left to right). (A) pEGFP-PKCα-WT–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (B) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the absence of TPA; (C) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (D) pEGFP-PKCα-A25E-K368M–transfected cells on H/120 in the presence of 5 ng/ml TPA. Bars, 50 μm.
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fig8: Effects of wild-type or dominant-negative PKCα overexpression on integrin-mediated migration. A375-SM cells were transiently transfected with various pEGFP-PKCα constructs and then tested for their ability to migrate into wounds on either a 50K substrate in the presence of soluble H/0 or an H/120 substrate. Still images were captured at the indicated times after wounding, and separate phase, EGFP, and merged images are shown (from left to right). (A) pEGFP-PKCα-WT–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (B) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the absence of TPA; (C) pEGFP-PKCα-A25E-K368M–transfected cells on 50K + H/0 in the presence of 5 ng/ml TPA; (D) pEGFP-PKCα-A25E-K368M–transfected cells on H/120 in the presence of 5 ng/ml TPA. Bars, 50 μm.
Mentions: Although BIM exhibits good specificity for PKC, we also attempted to perturb A375-SM migration using a dominant-negative approach. Mutant forms of PKC isoforms, tagged with GFP, were transfected into A375-SM transiently, and then their ability to close wounds was examined. Initially, wild-type pEGFP-PKCα (WT) and the double mutant pEGFP-PKCα-A25E-K368M were tested. A25E is a pseudosubstrate mutation that exposes the DAG/TPA-binding site and catalytic site. K368M is a mutation in the ATP-binding site that leaves the enzyme catalytically inactive. Thus, pEGFP-PKCα-A25E-K368M is inert, but TPA-responsive. On a 50K substrate in the presence of soluble H/0, transfection of pEGFP-PKCα-WT, either with (Fig. 8 A and Video 5) or without (unpublished data) TPA treatment 6 h after wounding, had no discernible effect on cell migration into the wound. When A375-SM cells transfected with dominant-negative pEGFP-PKCα-A25E-K368M were examined in the absence of TPA, migration again occurred normally (Fig. 8 B and Video 6). However, addition of TPA to recruit pEGFP-PKCα-A25E-K368M to the plasma membrane led to an almost complete blockade of cell movement (Fig. 8 C and Video 7). As shown most clearly in the video, GFP-expressing cells remained virtually motionless, exhibiting only occasional membrane ruffling and protrusion. However, nontransfected cells that lacked detectable GFP migrated past them and filled the wound. As an additional control, cells were transfected with pEGFP-PKCα-T497A, a substrate-binding mutant that can also act as a dominant-negative inhibitor. This mutant also prevented A375-SM cell migration on a 50K + H/0 substrate (unpublished data). To probe the contribution of PKCα to cell migration mediated by α4β1, pEGFP-PKCα-A25E-K368M–transfected cells were seeded on H/120 and wounded. In contrast to the findings on 50K + H/0, dominant-negative PKCα had no effect on wound closure (Fig. 8 D and Video 8), again suggesting that α4β1 does not depend on this enzyme.

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