Abstract
Growth factors, insulin, oxidative stress, and cytokines activate ERK1/2 by PKCδ and MEK1/2. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase and intracellular scaffold/bridge/anchor, is a nuclear transporter of MEK1/2-stimulated ERK1/2 (Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E., and Maines, M. D. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 6870-6875). hBVR, PKCδ, and MEK1/2 overlap in their tissue expression profile and type of activators. Presently, we report on formation of an hBVR-PKCδ-ERK2 ternary complex that is essential for ERK2 signal transduction and activation of genes linked to cell proliferation and cancer. MEK1/2 and the protein phosphatase PP2A were also present in the complex. When cells were stimulated with insulin-like growth factor-1 (IGF-1), an increased interaction between hBVR and PKCδ was detected by FRET-fluorescence lifetime imaging microscopy. hBVR and ERK2 were phosphorylated by PKCδ; however, the PKC was not a substrate for either ERK2 or hBVR. IGF-1 and phorbol ester increased hBVR/PKCδ binding; hBVR was required for the activation of PKCδ and its interaction with ERK2. The C-terminal phenylalanine residues of PKCδ (Phe(660), Phe(663), and Phe(665)) were necessary for binding to ERK2 but not for hBVR binding. Formation of the hBVR-PKCδ-ERK2 complex required the hBVR docking site for ERK, FXFP (DEF, C-box) and D(δ)-box (ILXXLXL) motifs. The hBVR-based peptide KKRILHCLGLA inhibited PKC activation and PKCδ/ERK2 interaction. Phorbol ester- and TNF-α-dependent activation of the ERK-regulated transcription factors Elk1 and NF-κB and expression of the iNOS gene were suppressed by hBVR siRNA; those activities were rescued by hBVR. The findings reveal the direct input of hBVR in PKCδ/ERK signaling and identify hBVR-based peptide regulators of ERK-mediated gene activation.
Highlights
ERK2 activation by PKC␦ relays cell growth signals. Human biliverdin reductase (hBVR) is a bridge/scaffold protein and nuclear transporter of ERK
Complex Is Formed between ERK2, PKC␦, and hBVR—Because hBVR was previously shown to form a complex with ERK1/2 [7] and presently found to bind PKC␦, we examined whether a complex is formed between the three proteins
Activation loop in proximity to its activator kinase MEK; in the nucleus, the activated ERK1/2 is brought in contact with its substrate Elk1 by hBVR; those functions of the reductase are independent of its kinase activity
Summary
ERK2 activation by PKC␦ relays cell growth signals. hBVR is a bridge/scaffold protein and nuclear transporter of ERK. Functions include Ser/Thr/Tyr kinase activity, intracellular transport of signaling molecules, regulation of stress-responsive gene expression, and cytoplasm-cell membrane translocation of PKCII and PKC, members of the conventional and atypical PKCs, respectively, as well as cytoplasm-nuclear transport of hematin and, as noted above, ERK1/2 [7, 13,14,15]. The study has revealed formation of a complex among hBVR, PKC␦, and ERK2 that is required for activation of ERK2 In this complex, hBVR functions as a bridge rather than a kinase, with the two specific sequences in the protein constituting the contact points between hBVR and its partners. Because in tumor cells proliferation is stimulated by deregulated ERK [26], the present observation that small hBVR-based peptides can effectively block activation of ERK2 by PKC␦ offers a potentially viable prospect for development of therapeutic agents to control tumor growth in a variety of malignancies
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