Abstract
Activation of the canonical mitogen-activated protein kinase (MAPK) cascade by soluble mitogens is blocked in non-adherent cells. It is also blocked in cells in which the cAMP-dependent protein kinase (PKA) is activated. Here we show that inhibition of PKA allows anchorage-independent stimulation of the MAPK cascade by growth factors. This effect is transient, and its duration correlates with sustained tyrosine phosphorylation of paxillin and focal-adhesion kinase (FAK) in non-adherent cells. The effect is sensitive to cytochalasin D, implicating the actin cytoskeleton as an important factor in mediating this anchorage-independent signalling. Interestingly, constitutively active p21-activated kinase (PAK) also allows anchorage-independent MAPK signalling. Furthermore, PKA negatively regulates PAK in vivo, and whereas the induction of anchorage-independent signaling resulting from PKA suppression is blocked by dominant negative PAK, it is markedly prolonged by constitutively active PAK. These observations indicate that PKA and PAK are important regulators of anchorage-dependent signal transduction.
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Acknowledgements
We thank D. Howe and K. McCarthy (UNC) and R. Maurer (Oregon Health Sciences University) for creating and providing the stable cell lines and various PKA constructs used in this study. We also thank M. Cobb (Southwestern Medical Center) and G. Bokoch (Scripps Research Institute) for PAK constructs, J. Sondek (UNC) for purified Cdc42, and S. Short, A. Aplin and L. Graves for helpful discussions. This work was supported by grants from the National Institutes of Health (to R.L.J.) and the American Cancer Society (to A.K.H.).
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Howe, A., Juliano, R. Regulation of anchorage-dependent signal transduction by protein kinase A and p21-activated kinase. Nat Cell Biol 2, 593–600 (2000). https://doi.org/10.1038/35023536
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DOI: https://doi.org/10.1038/35023536
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