Direct Interaction between Ras and the Kinase Domain of Mitogen-activated Protein Kinase Kinase Kinase (MEKK1)

Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKcooH) binds to GST-Rasv12 in a GTP-dependent manner. Purified bacterially expressed MEKKcooH binds to GST-Rasv12(GTPγS) (GTPγS is guanosine 5’-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKcooH to GST-Rasv12(GTPγS). MEKKcooH complexed with GST-Rasv12(GTPγS) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras•GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families. Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKcooH) binds to GST-Rasv12 in a GTP-dependent manner. Purified bacterially expressed MEKKcooH binds to GST-Rasv12(GTPγS) (GTPγS is guanosine 5’-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKcooH to GST-Rasv12(GTPγS). MEKKcooH complexed with GST-Rasv12(GTPγS) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras•GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.