Mortalin Modulates MEK/ERK Activity by Regulating the Physical Interaction between MEK1/2 and Protein Phosphatase 1 Alpha

Aberrant Raf/MEK/ERK signaling is a central feature in many human malignancies. This hyperactivated oncogenic signal is mainly driven by constitutive activating mutations in receptor tyrosine kinase, Ras or B‐Ras oncogenes. Although precision medicine, such as highly selective small molecule kinase inhibitors, has been developed to treat cancer patients by targeting multiple members along the signaling axis, variable efficacy and success has been observed in clinical outcomes. This is mainly due to the rapidly developed drug resistance resulting insensitivity to treatment and tumor relapse begging the need for alternative therapeutic strategy. Growing body of scientific evidence suggests that sustained activity of Raf/MEK/ERK signaling can also trigger tumor suppressive response by inducing growth arrest and cell death in many cell types (Reviewed in Ref. 1), indicating the presence of a tumor defense mechanism that must be inactivated for carcinogenesis to occur. Our previous study identified mortalin/HSPA9, a member of the heat shock protein 70 family, as a novel MEK1/2‐interacting protein by proteomic screening. Mortalin is a crucial regulator that determines the physiological outputs of Raf/MEK/ERK signaling in Ras/Raf‐driven cancers proposing mortalin as a novel therapeutic target selective to these tumors (Ref. 2). However, the molecular mechanism underlying the regulatory role of mortalin on MEK/ERK activity is unclear. In the present study, using in vitro dephosphorylation assay we discovered that inhibiting protein phosphatase 1 upregulates MEK1/2 phosphorylation in B‐Raf V600E ‐mutated human melanoma and K‐Ras G12D/V ‐mutated pancreatic cancer cells. Intriguingly, mortalin depletion had no effect on cellular levels of B‐Raf, K‐Ras, or PP1α protein and its regulatory phosphorylation indicating the protein levels of upstream kinase or protein phosphatases are not a target for regulation. Indeed, co‐immunoprecipitation and in vitro binding assay showed that mortalin physically interacts with PP1α and MEK1/2, and facilitates PP1α‐MEK1/2 complex formation. Binding between mortalin and PP1α, and MEK is sensitive to the presence of ATP and ADP suggesting a chaperone‐client relationship. Importantly, the valine 482 and QGE tripeptide in the groove region and the substrate lid of peptide binding domain are key regions for the regulation. Our data suggest that mortalin, which processes novel non‐chaperone function as a MEK/ERK signaling modulator, attenuates MEK1/2 phosphorylation by promoting their physical interactions with PP1α. Expression levels of PP1α are upregulated in human melanoma and pancreatic cancer specimens demonstrating its significance in tumorigenesis. Our results reveal a mechanistic rationale to develop selective mortalin inhibitor which might be a potential therapeutic strategy for Ras/Raf‐driven cancers. Support or Funding Information This work was supported by National Cancer Institute (R01CA138441) and American Cancer Society (RSGM‐10‐189‐01‐TBE) to J.PMortalin is a novel regulator to modulate oncogenic Raf/MEK/ERK signaling