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Formation of a Ternary Complex of Human Biliverdin Reductase/ PKC-δ/ ERK2

Image of FRET-FLIM

Association of EGFP-tagged PKC-δ and DsRed2-tagged hBVR in IGF-1
treated cells as determined by confocal microscopy (a) and Fluorescence
Resonance Energy Transfer coupled with Fluorescence Lifetime
Imaging Microscopy (b,c).

We have shown for the first time that human biliverdin reductase (hBVR) forms a macromolecular complex with protein kinase Cδ and the extracellular receptor kinase, ERK2; and that formation of this complex is required for downstream nuclear signaling mediated by ERK2. The ERK proteins are of fundamental importance in the regulation of cell proliferation and differentiation, and of the stress responses. hBVR and PKCδ were observed to interact upon stimulation by insulin-like growth factor-1 (IGF-1) by various approaches including FRET in live cells, as detected by fluorescence lifetime imaging microscopy FLIM. It was demonstrated that the hBVR-ERK2-PKCδ complex includes MEK, suggesting the assembly of an elaborate, hBVR-centered signal transduction complex. In the complex, hBVR functions primarily as a scaffold/bridge/anchor protein. The hBVR-anchored complex results in activation of both PKCδ and of ERK. That the protein is essential for activation of ERK2 by PKCδ was suggested by finding that siRNA-mediated reduced levels of hBVR expression resulted in a severe attenuation of PKCδ signaling; the inhibition was rescued by expression of hBVR from a plasmid. Because unrestrained activation of the signaling complex may not be the best long-term strategy for the cell, it is noteworthy that at least one protein phosphatase, PP2A, that targets PKCδ, was also present in the complex. This suggests that the complex carries within it the means of its self-regulation. Previous studies from this laboratory had also shown that a ternary complex is formed between hBVR, ERK1/2 and MEK1.

Finding that the both kinases upstream of ERK are dependent on the hBVR scaffolding function defines hBVR as an essential partner for signaling activity of the ERK kinases that constitute one of the three major branches of MAPK signal transduction pathways. The significance of the findings is underscored by the essential role of ERK and PKCδ in a wide spectrum of cellular functions. Currently, some fifty transcription factors and proteins involved in the cellular stress response are believed to be activated by ERK1/2. The list includes Elk1, NF-κB and iNOS, that were shown in the Gibbs et al. manuscript to be affected by hBVR-based peptides that, in the course of the study, were found to have a profound effect on assembly of the hBVR-PKCδ-ERK2 complex. ERK kinases are a major players in regulation of cell growth, differentiation and division. Cancer cells display uncontrolled cell division and growth, prompting invasion and consequent destruction of neighboring tissues. 30% of tumors have aberrant ERK activation. The pathway is ordinarily a carefully orchestrated series of events that initiates at the cell membrane, and ultimately results in gene activation. Moreover, the ERK2 signaling pathway has profound effects on the sensitivity of cells to chemotherapeutic drugs; downregulation of the ERK pathway reduces the sensitivity. Therefore, targeting the PKCδ/ERK and MEK/ERK pathways may well be an effective approach for therapeutic intervention in drug resistant cancers that are linked to mutations in signaling pathways. Although the pathways leading to drug resistance are not as of yet completely elucidated, a major focus of pharmaceutical research is in the development of inhibitors of ERK activation. It is also noted that for ERK to translocate into and out of the nucleus it requires a carrier protein with a nuclear localization signal (NLS) sequence and a nuclear export signal (NES). The nuclear import function is fulfilled by hBVR and Gab1; however, the only protein described thus far as a carrier for exporting ERK1/2 from the nucleus is hBVR.

Proposed mechanism for hBVR, PKC-δ and ERK2 ternary complex formation

Proposed mechanism for hBVR, PKC-δ and ERK2 ternary complex formation.

Altered activity of PKCδ has been implicated in a variety of disorders, including breast cancer, atherosclerosis, Parkinson's disease, as well as drug resistance. PKCδ is upregulated by estrogen. Development of resistance to drugs such as tamoxifen used in treatment of hormone-responsive breast tumors is regulated directly by PKCδ and enhanced by over-expression of the kinase. Unregulated growth of smooth muscle cells is a precipitating event in atherosclerosis. Nuclear transport of the catalytic domain of PKCδ accelerates the apoptosis program by inhibition of DNA repair functions, which is a major factor in the development of Parkinson's disease that is associated with death of dopaminergic neurons. Conversely, PKCδ may be cytoprotective in retinopathy associated with type-2 diabetes. On balance, it is apparent that PKCδ regulation is critical to health, and that over- or under-expression is likely to be detrimental. Restoration of normal levels of activity of this kinase is, therefore, likely to be importance in controlling disease.

Thus, we have introduced a new dimension to the activation of ERK by PKCδ and MEK, by examining the role of hBVR in activation of its upstream kinases, building on our earlier work with activation of ERK directly by hBVR, and the activation of other PKCs.

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