Studies on Extracellular Signal-Regulated Kinase-1/2 (ERK1/2) Function and Identification of Small Molecule Inhibitors that Selectively Target ERK1/2 Regulation of Activator Protein-1 (AP-1) Function in Cancer Cell Proliferation

Abstract

Excessive activation of the Raf/MEK/ERK pathway participates in the pathogenesis of numerous human tumors. In this pathway, extracellular signal regulated kinase 1/2 (ERK1/2) occupy a unique position on which signals from various membrane receptors converge. Therefore, ERK1/2 have been appealing targets for the development of anticancer drugs. In the recent years, careful examination of ATP binding site of protein kinases coupled with structure-based drug design methods has largely facilitated development of specific protein kinase inhibitors. The crystal structures of ERK2 with ATP complex refined to 2.3 Å have been reported previously. However, the details of interactions between ERK2 and ATP binding pocket were not certain because of low resolution. We have obtained high resolution crystals and refined the structures of the apoenzyme, ATP-bound ERK2, ADP-bound ERK2 to 1.8, 1.7 and 1.8 Å respectively. We then described the identification of a novel lead compound with a thienyl benzenesulfonate scaffold that targets the F-domain Recruitment site (FRS) of ERK1/2. Our crystal structure data suggested that this compound interacted with ERK2 in the vicinity of the FRS. Biological analyses further showed that this compound, along with a few structurally similar analogs derived from computational methods based on information provided by the crystal structure, preferentially inhibited F-site containing substrates that form the activator protein-1 (AP-1) transcription factor complex including c-Fos, Fra1, and FosB. Cell viability analysis revealed that melanoma cell lines that harbor constitutively activated ERK1/2 driven by activating B-Raf mutants were more sensitive to growth inhibition by these compounds. Lastly, we discovered a novel mechanism by which ERK1/2 activation regulates the phosphorylation of protein kinase C (PKC) isoforms PKCβII/δ. Our studies demonstrated that growth factor mediated ERK1/2 activation regulated PKCβII/δ activity as inhibition of ERK1/2 affected PKC substrate phosphorylation. Moreover, we provide evidence that inhibition of ERK1/2 reduced cell migration through a mechanism involving regulation of PKCβII. Thus, these data indicate that ERK1/2 proteins act as upstream regulators of PKC proteins and that inhibition of this regulation may have utility in preventing cancer cell migration and metastasis.