Friedegund Meier ¹, Birgit Schittek ¹, Silke Busch ¹, Claus Garbe ¹, Keiran Smalley ², Kapaettu Satyamoorthy ², Gang Li ², and Meenhard Herlyn ²

1 Department of Dermatology, University of Tuebingen, Tuebingen, Germany, ² Wistar Institute, Philadelphia, Pennsylvania 19104

FIGURES

Figure 1. Schematic representation of the Ras-Raf-MEK-ERK (MAPK) signaling pathway. An extracellular factor such as a growth factor (GF) interacts with its receptor tyrosine kinase (RTK) and induces receptor dimerisation and autophosphorylation on tyrosine residues. The phosphotyrosines function as docking sites for the growth-factor-receptor-bound protein 2 adapter protein (Grb2). Grb2 pulls the GDP/GTP exchange factor son of sevenless (SOS) to the cell membrane. SOS induces switching of the Ras-family GTPases from the inactive GDP-bound state to the active GTP-bound state. Activated Ras binds to the Raf serine/threonine kinases (A-Raf, B-Raf, C-Raf/Raf-1) and recruits them to the cell membrane. Activation of B-Raf is obtained after binding to Ras alone whereas for activation of A-Raf and Raf-1 additional signals are required. Raf-1 activation is a multi-step process that requires the phosphorylation of activating sites by other kinases (e.g. Src) as well as the dephosphorylation of inhibitory sites by protein phosphatase 2A (not shown). Activated Raf phosphorylates and activates MEK which phosphorylates and activates ERK. The Raf-MEK-ERK cascade is scaffolded by the kinase suppressor of Ras (not shown). Activated ERK has many substrates in the cytosol and can also enter the nucleus to regulate gene expression by phosphorylating transcription factors (TFs).

Figure 2. Activation of Ras-Raf-MEK-ERK (MAPK) signaling pathway in melanoma occurs through multiple mechanisms: activating mutations of N-Ras or B-Raf, autocrine and paracrine growth factors, and adhesion receptor signaling.

Figure 3. Role of Ras-Raf-MEK-ERK (MAPK) signaling pathway in melanoma invasion. Constitutive activation of MAPK signaling pathway appears to be associated with (1) downregulated E-cadherin expression freeing melanoma cells from keratinocyte-mediated control, (2) upregulated integrin expression promoting survival and invasive growth of melanoma cells in ECM and (3) increased expression and activity of proteolytic enzymes facilitating degradation of ECM.

Figure 4. Schematic representation of the PI3K-AKT (AKT) signaling pathway (57). An extracellular factor such as a growth factor interacts with its receptor protein tyrosine kinase (RPTK) resulting in autophosphorylation of tyrosine residues. Phosphatidylinositol-3 kinase (PI3K) consisting of an adaptor subunit p85 and a catalytic subunit p110 is translocated to the cell membrane and binds to phosphotyrosine consensus residues of the RPTK through ist its adaptor subunit. This results in allosteric activation of the catalytic subunit leading to production of phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 recruits signaling proteins with pleckstrin homolgy (PH) domains to the cell membrane including AKT. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is a PIP3 phosphatase and negatively regulates the PI3K-AKT pathway. The interaction of PIP3 with the PH domain of AKT likely induces conformational changes in AKT, thereby exposing the two main phosphorylation sites at T308 and S473. T308 and S473 phosphorylation by protein serine/threonine kinase 3'-phosphoinositide-dependent kinases 1 and 2 (PDK1 and PDK2) is required for maximal AKT activation. Activated AKT translocates to the nucleus and mediates the activation and inhibition of various targets resulting in cellular survival and cell growth and proliferation.

Figure 5. Activation of PI3K-AKT (AKT) signaling pathway in melanoma occurs through loss of PTEN, a negative regulator for PI3K-induced signaling, autocrine and paracrine growth factors, and adhesion receptor signaling.