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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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Isoform specificity of the effects of dominant-negative PKCα on α5β1-dependent migration. A375-SM cells were transiently transfected with various pEGFP-PKC constructs and then tested for their ability to migrate into wounds on a 50K substrate in the presence of soluble H/0. 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-PKCδ-K376M–transfected cells in the presence of 5 ng/ml TPA; (B) pEGFP-PKCζ-K281M–transfected cells in the absence of TPA; (C) pEGFP-PKCδ-WT in the presence of 5 ng/ml TPA. Bars, 50 μm.
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fig9: Isoform specificity of the effects of dominant-negative PKCα on α5β1-dependent migration. A375-SM cells were transiently transfected with various pEGFP-PKC constructs and then tested for their ability to migrate into wounds on a 50K substrate in the presence of soluble H/0. 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-PKCδ-K376M–transfected cells in the presence of 5 ng/ml TPA; (B) pEGFP-PKCζ-K281M–transfected cells in the absence of TPA; (C) pEGFP-PKCδ-WT in the presence of 5 ng/ml TPA. Bars, 50 μm.

Mentions: Because dominant-negative PKCα might also block the activity of other PKC isoforms by competition for substrates, we performed control experiments with dominant-negative mutants of PKCδ and PKCζ. As shown in Fig. 9 (A and B) and Videos 9 and 10, transfection with either dominant-negative pEGFP-PKCδ-K376M and pEGFP-PKCζ-K281M, with or without TPA treatment, did not reduce A375-SM migration on 50K + H/0. This suggests that the dominant-negative effects of pEGFP-PKCα-A25E-K368M are specific to the α isoform. Interestingly, transfection of wild-type pEGFP-PKCδ, and treatment with TPA, led to a suppression of migration (Fig. 9 C and Video 11). This result is consistent with the previously reported syndecan-4–dependent inhibitory effect of PKCδ on PKCα (Murakami et al., 2002). Together, these results demonstrate that migration mediated by α4β1 and α5β1 exhibits the same differential dependency on PKCα activity as focal adhesion formation.


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)

Isoform specificity of the effects of dominant-negative PKCα on α5β1-dependent migration. A375-SM cells were transiently transfected with various pEGFP-PKC constructs and then tested for their ability to migrate into wounds on a 50K substrate in the presence of soluble H/0. 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-PKCδ-K376M–transfected cells in the presence of 5 ng/ml TPA; (B) pEGFP-PKCζ-K281M–transfected cells in the absence of TPA; (C) pEGFP-PKCδ-WT in the presence of 5 ng/ml TPA. Bars, 50 μm.
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Related In: Results  -  Collection

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fig9: Isoform specificity of the effects of dominant-negative PKCα on α5β1-dependent migration. A375-SM cells were transiently transfected with various pEGFP-PKC constructs and then tested for their ability to migrate into wounds on a 50K substrate in the presence of soluble H/0. 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-PKCδ-K376M–transfected cells in the presence of 5 ng/ml TPA; (B) pEGFP-PKCζ-K281M–transfected cells in the absence of TPA; (C) pEGFP-PKCδ-WT in the presence of 5 ng/ml TPA. Bars, 50 μm.
Mentions: Because dominant-negative PKCα might also block the activity of other PKC isoforms by competition for substrates, we performed control experiments with dominant-negative mutants of PKCδ and PKCζ. As shown in Fig. 9 (A and B) and Videos 9 and 10, transfection with either dominant-negative pEGFP-PKCδ-K376M and pEGFP-PKCζ-K281M, with or without TPA treatment, did not reduce A375-SM migration on 50K + H/0. This suggests that the dominant-negative effects of pEGFP-PKCα-A25E-K368M are specific to the α isoform. Interestingly, transfection of wild-type pEGFP-PKCδ, and treatment with TPA, led to a suppression of migration (Fig. 9 C and Video 11). This result is consistent with the previously reported syndecan-4–dependent inhibitory effect of PKCδ on PKCα (Murakami et al., 2002). Together, these results demonstrate that migration mediated by α4β1 and α5β1 exhibits the same differential dependency on PKCα activity as focal adhesion formation.

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