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Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1)
M Russell1, C A Lange-Carter, G L Johnson
1Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
Abstract:
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(GTP gamma S) (GTP gamma S 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(GTP gamma S). MEKKCOOH complexed with GST-RasV12(GTP gamma S) 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.
Insights
Mitogen-activated protein kinase kinase kinase 1 (MEKK1) directly binds Ras.GTP, a key signaling molecule. This interaction reveals a new link between Ras and protein kinases, impacting cellular pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase involved in stress-activated and mitogen-activated protein kinase pathways.
- MEKK1 activation is triggered by growth factors and activated Ras expression.
Purpose of the Study:
- To investigate the direct interaction between MEKK1 and Ras.
- To elucidate the functional consequences of MEKK1-Ras binding on kinase activity.
Main Methods:
- Utilized bacterial expression to purify the kinase domain of MEKK1 (MEKKCOOH).
- Performed glutathione S-transferase (GST) pull-down assays with GST-RasV12 and GTP gamma S to demonstrate direct protein binding.
- Employed a Ras effector domain peptide to assess binding specificity.
- Assayed the kinase activity of MEKKCOOH in complex with Ras.
Main Results:
- The kinase domain of MEKK1 (MEKKCOOH) directly binds to GTP-bound Ras (GST-RasV12(GTP gamma S)) in a GTP-dependent manner.
- A Ras effector domain peptide inhibited the binding of MEKKCOOH to GST-RasV12(GTP gamma S), confirming specificity.
- The MEKKCOOH-Ras.GTP complex demonstrated the ability to phosphorylate MEK1, a downstream kinase.
Conclusions:
- MEKK1 directly interacts with Ras.GTP.
- Ras engages with protein kinases from both the Raf and MEKK families, expanding our understanding of Ras signaling networks.
- This direct interaction suggests a novel regulatory mechanism in cellular signaling pathways controlled by Ras.