Abstract
PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser(230) and Ser(237)), flanking the ATP-binding motif (Ser(21)) and in PHPS sequence (Ser(33)) as targets of PKCδ. Ser(21) and Ser(230) were also phosphorylated in hBVR-based peptides. The Ser(230)-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.
Highlights
We examined the consequence of the Human biliverdin reductase (hBVR)/ PKC␦ interaction on hBVR phosphorylation, aiming to identify specific targets of the PKC by evaluating several candidate phosphorylation sites on hBVR that are contained within the consensus phosphorylation motifs of PKC␦
HBVR Reductase Activity Is Increased in the Presence of PKC␦, and Biliverdin Blocks Activation of PKC␦ by phorbol 12-myristate 13-acetate (PMA) in Cells— Having established that hBVR is a substrate for PKC␦ [7], we examined the consequences of phosphorylation by PKC␦ on the reductase activity of the enzyme; as noted earlier, this activity is dependent on hBVR phosphorylation [5]. hBVR was used as a substrate by PKC␦ in vitro
HBVR is an activator of PKC␦ [7], its substrate, biliverdin, and two peptides, designed based on the primary structure of the hBVR protein, are potent inhibitors of the PKC
Summary
Materials—Recombinant activated PKC␦ for in vitro studies, TNF-␣ and PMA, were obtained from Calbiochem. Mass Spectrometry Analysis of Peptides from Tissue Culture— Cells were transfected with pcDNA-PKC␦ and serum-starved as described above They were treated with 100 nM PMA for 15 min, and the in situ PKC␦ assay [42] was used to introduce the peptides into the cells. Cells were washed and incubated for 10 min at 30 °C in 50 l of kinase assay buffer (137 mM NaCl, 5.4 mM KCl, 10 mM MgCl2, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 25 mM -glycerophosphate, 5.5 mM D-glucose, 5 mM EGTA, 1 mM CaCl2, 20 mM HEPES, pH 7.2, 50 g/ml digitonin, 120 g/ml PKC␦ peptide substrate, and 50 M ATP). Kinetic data for the peptide substrate were fitted to the Michaelis-Menten equation using Prism 3.0 software (GraphPad, San Diego)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
