Protein Kinase C-βII Represses Hepatocyte Growth Factor-Induced Invasion by Preventing the Association of Adapter Protein Gab1 and Phosphatidylinositol.

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Protein Kinase C-βII Represses Hepatocyte Growth Factor-Induced Invasion by Preventing the Association of Adapter Protein Gab1 and Phosphatidylinositol 3-Kinase in Melanoma Cells Masahiro Oka, Ushio Kikkawa, Chikako Nishigori Journal of Investigative Dermatology Volume 128, Issue 1, Pages 188-195 (January 2008) DOI: 10.1038/sj.jid.5700961 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 HGF-induced activation of c-Met in melanocytes and melanoma cells. The cells were treated with 100ng/ml HGF for the indicated time. Cell lysates were immunoprecipitated (IP) with anti-phosphotyrosine antibody PY20 followed by immunoblot (IB) analysis using the anti-c-Met antibody. The results are representative of three independent experiments. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Activation of PI3K in melanocytes and melanoma cells. (a) Melanocytes were treated with either 100ng/ml HGF (left panels) or with 5μg/ml insulin (right panels) for the indicated time. The immunoprecipitates with PY20 from cell lysates were assayed for PI3K activity. (b) Melanocytes and melanoma cells were treated with 100ng/ml HGF for the indicated time (upper panel) or treated with various concentrations of HGF for 5minutes (lower panel). The relative radioactivities of PI(3)P are expressed as a percentage of that in untreated control cells, and data are shown as means±SD from three independent experiments. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Activation of ERK in melanocytes and melanoma cells. The cells were treated with 100ng/ml HGF for the indicated time. Cell lysates were subjected to immunoblot analysis using anti-phospho-ERK and anti-ERK antibodies, respectively. The results shown are representative of three independent experiments. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The electrophoretic mobility of Gab1. Gab1 immunoprecipitated from melanocytes and melanoma cells was subjected to immunoblot analysis using an antibody against Gab1 (upper panels). MeWo cells were infected with each adenovirus vector, and the immunoprecipitated Gab1 and cell lysates were subjected to immunoblot analysis using anti-Gab1 anti-PKC antibodies, respectively (lower panels). Where indicated, the immunoprecipitates were treated with calf intestine alkaline phosphatase in vitro. Open and closed arrows indicate the position of Gab1. The results shown are representative of three independent experiments. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Association of the p85 subunit of PI3K and Gab1 in MeWo cells but not melanocytes. MeWo cells infected with either AxPKC-β or AxLacZ and melanocytes were serum-starved and treated with 100ng/ml HGF for the indicated time. (a) Gab1 in MeWo cells (upper and middle panels) and in melanocytes (lower panel) was immunoprecipitated from cell lysates and subjected to immunoblot analysis using anti-Gab1 and anti-p85 subunit, respectively. (b) Gab1 in MeWo cells was immunoprecipitated from cell lysates and subjected to immunoblot analysis using anti-Gab1 and PY20 antibodies, respectively. The results shown are representative of three independent experiments. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 PI3K activation and invasive activity of melanoma cells. The cells were infected with each adenovirus vector and serum-starved. PI3K activity was measured after treatment with 100ng/ml HGF for 5minutes, and is expressed as a percentage of that in untreated cells (upper panels). Data are shown as means±SD from three independent experiments. In vitro invasion assays were carried out and the results are expressed as a percentage of that in control uninfected cells (lower panels). Data are shown as means±SD (n=3). The results shown are representative of three independent experiments. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 HGF-induced signaling pathways in the melanocytes and melanoma cells. The regulatory mechanisms revealed in this study are schematically presented. Journal of Investigative Dermatology 2008 128, 188-195DOI: (10.1038/sj.jid.5700961) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions