The G-protein-coupled receptor kinase 5 inhibits NF␬B transcriptional activity by inducing nuclear accumulation of I␬B␣ Daniela Sorrientoa, Michele Ciccarellia, Gaetano Santullia, Alfonso Campanilea, Giovanna Giuseppina Altobellia, Vincenzo Ciminib, Gennaro Galassoa, Dalila Astonec, Federico Piscionea, Lucio Pastorec, Bruno Trimarcoa, and Guido Iaccarinoa,1 Dipartimento di aMedicina Clinica, Scienze Cardiovascolari, ed Immunologiche, and bScienze Biomorfologiche e Funzionali, Universita`Federico II, 80134 Naples, Italy; and cCentro di Ingegneria Genetica –Biotecnologie Avanzate, 80145 Naples, Italy Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved September 12, 2008 (received for review May 12, 2008) G-protein-coupled receptor (GPCR) kinases, GRKs, are known as the nucleus and to bind to specific sequences in the promoter or serine/threonine kinases that regulate GPCR signaling, but recent enhancer regions of target genes (11). Recently this view has been findings propose functions for these kinases besides receptor desen- partially revisited by the description of NES and NLS sequences on sitization. Indeed, GRK5 can translocate to the nucleus by means of a the NF␬B/I␬B␣ complex that allow a continuous shuttling between nuclear localization sequence, suggesting that this kinase regulates the nucleus and the cytosol by means of nuclear transporters, such transcription events in the nucleus. To evaluate the effect of GRK5– as CRM1 (1, 3). New partners of this complex are described along I␬B␣ interaction on NF␬B signaling, we induced the overexpression the way: ␤-arrestin 2 interacts with the NF␬B/I␬B␣ complex within and the knockdown of GRK5 in cell cultures. GRK5 overexpression the cytosol, causing the blockade of NF␬B transcriptional activity causes nuclear accumulation of I␬B␣, leading to the inhibition of NF␬B (16, 17). Studying a NF␬B cognate precursor—NF␬B p105, which transcriptional activity. Opposite results are achieved by GRK5 knock- actively participates in cytosolic signal transduction but lacks tran- down through siRNA. A physical interaction between GRK5 and I␬B␣, scriptional activity—Parameswaran et al. (18) recently described the rather than phosphorylative events, appears as the underlying mech- ability of GRK5 to physically interact with NF␬B p105. These anism. We identify the regulator of gene protein signaling homology authors did not investigate the eventual effects of GRK5 on NF␬B domain of GRK5 (RH) and the N-terminal domain of I␬B␣ as the transcriptional activity. regions involved in such interaction. To confirm the biological rele- NF␬B drives VEGF, bFGF, IL-8, and other cytokines’ expres- vance of this mechanism of regulation for NF␬B, we evaluated the sions (19). In the endothelium, cytokine production sustains tissue effects of GRK5-RH on NF␬B-dependent phenotypes. In particular, regeneration by providing the start to neoangiogenesis and there- GRK5-RH overexpression impairs apoptosis protection and cytokine fore blood and nutrient support. Failure of NF␬B activation is production in vitro and inflammation and tissue regeneration in vivo. associated with impaired cytokine production and angiogenesis Our results reveal an unexpected role for GRK5 in the regulation of (20, 21). NF␬B transcription activity. Placing these findings in perspective, this On the basis of these preexisting observations, we hypothesized mechanism may represent a therapeutic target for all those condi- that GRK5 could regulate NF␬B transcriptional activity. In par- tions involving excessive NF␬B activity. ticular, we investigated whether GRK5 affects I␬B␣ cellular levels and NF␬B activity and the molecular basis for GRK5 and I␬B␣ angiogenesis ͉ gene transcription ͉ inflammation ͉ signal transduction physical interaction. Also, we assessed the effects on NF␬B- dependent phenotypes such as apoptosis, cytokine production, and -protein-coupled receptor (GPCR) kinases, GRKs, constitute in vivo and in vitro angiogenesis. a large family of serine/threonine protein kinases that regulate G Results GPCR signaling (1–3), consisting of 7 isoforms that share structural and functional similarities (4). A central catalytic domain is flanked GRK5 Overexpression Causes I␬B␣ Nuclear Accumulation. We evalu- ␬ ␣ by an N-terminal domain that includes a region of homology to ated GRK5 and I B cellular localization by Western blot in bovine regulators of G-protein signaling (RH) and a C-terminal domain of aorta endothelial cells (BAEC) overexpressing the WT bovine ␬ ␣ variable length (5, 6). The catalytic domain of GRKs is relatively GRK5 gene. The overexpression of GRK5-WT increases I B ␬ ␣ well-conserved among the members of different subfamilies levels in whole-cell extracts by inducing I B nuclear accumulation (Ϸ45% sequence identity), whereas N-terminal RH domains dis- and does not change cytosol levels (Fig. 1A). Fig. 1B shows that the play weak homology (Ϸ27%) and C termini have little or no total and nuclear levels of GRK5 after transfection increase in a sequence homology. GRKs have different tissue distribution, sub- time-dependent manner and are associated with a progressive ␬ ␣ cellular localization, and kinase activity regulation (7, 8). GRKs accumulation of I B levels both in whole-cell lysates and nuclear ␬ mostly localize at the plasma membrane (2), but recently Johnson extracts. Accordingly, NF B activity decreases over time, synchro- ␬ ␣ et al. (7) demonstrated that GRK4–6 (but not other GRKs) can nized with the increase of I B levels. These results suggest that shuttle between cytosol and nucleus through functional nuclear localization sequence (NLS) and nuclear exporting sequence Author Contributions: G.I. designed research; D.S., M.C., G.S., A.C., G.G.A., V.C., G.G., D.A., (NES), thus suggesting a nuclear effect for the GRK4–6 subfamily. F.P., and L.P. performed research; D.S., B.T., and G.I. analyzed data; and D.S., B.T., and G.I. NF␬B is an ubiquitously expressed and highly regulated dimeric wrote the paper. transcription factor (3) regulating the expression of genes respon- The authors declare no conflict of interest. sible for innate and adaptive immunity, tissue regeneration, stress This article is a PNAS Direct Submission. responses, apoptosis, cell proliferation, and differentiation (9–14). 1 ␬ To whom correspondence should be addressed at: Medicina Clinica, Scienze Cardiovascolari Within the canonical view of the regulation of the NF B activity, ed Immunologiche, Federico II University, Via Pansini 5; Edificio 2, 80131 Naples, Italy. E-mail: the inactive NF␬B/I␬B␣ complex localizes in the cytosol until an [email protected]. extracellular stimulus, such as TNF␣ or LPS, induces I␬B kinase This article contains supporting information online at www.pnas.org/cgi/content/full/ (IKK)-mediated I␬B␣ phosphorylation and subsequent degrada- 0804446105/DCSupplemental. tion by the proteasome (15). NF␬B is therefore free to shuttle into © 2008 by The National Academy of Sciences of the USA 17818–17823 ͉ PNAS ͉ November 18, 2008 ͉ vol. 105 ͉ no. 46 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804446105 Downloaded by guest on October 1, 2021 B BAEC BAEC C BAEC whole cell lysates nuclear extracts whole cell lysates GRK5 0 0.5 1 2 ug GRK5 0 12 24 36 48 0 12 24 36 48 hrs GRK5 GRK5 GRK5 I B A BAEC I B I B Actin H3 Actin GRK5 WT - + - + - + 450 GRK5 450 400 400 I B 350 350 300 300 B activity Actin B activity 250 250 H3 200 200 Whole Cytosol Nucleus 150 150 100 100 50 50 Relative NF Relative NF Relative D HEK293 0 0 whole cell lysates 0 12 24 36 48 hrs 0 0.5 1 2 GRK5 ( g) CTRL GRK5 GRK5 HEK293 BAEC WT siRNA E F whole cell lysates nuclear extracts GRK5 CTRL GRK5 GRK5 GRK5 0 5 10 20 ug WT K215R I B siRNA GRK5 GRK5 Actin I B I B 800 700 Actin NF B 600 H3 500 B activity 800 400 1200 700 300 1000 600 200 800 500 B activity B Relative NF 100 B activity 400 600 0 300 CTRL GRK5 GRK5 400 200 WT siRNA 200 Relative NF 100 Relative NF 0 0 CTRL GRK5 GRK2 0 5 10 20 siRNA ( g) GRK5 WT K215R Fig. 1. GRK5 binds and stabilizes I␬B␣, causing inhibition of NF␬B activity. (A)I␬B␣ and GRK5 levels were analyzed in whole, cytosolic, and nuclear extracts by Western blot in BAEC overexpressing GRK5-WT. The overexpression of GRK5-WT increases I␬B␣ levels in whole-cell extracts and induces I␬B␣ nuclear accumulation. (B)To evaluate the effects of GRK5 on I␬B␣ turnover, we analyzed GRK5 and I␬B␣ expression in a time-course of GRK5 transfection. The increase of GRK5 levels induces a progressive increase of I␬B␣ levels and a reduction of NF␬B activity (*P Ͻ 0.05 vs. control). (C) A time course of transfection of GRK5 was performed in BAEC and GRK5 and I␬B␣ levels analyzed both in whole- and nuclear-cell extracts. The time-dependent increase of GRK5 levels associates to I␬B␣ stabilization and nuclear localization and subsequently NF␬B activity inhibition (*P Ͻ 0.05 vs. control). (D) HEK293 transfected with GRK5-WT or GRK5siRNA were analyzed by WB for GRK5 and I␬B␣ expression. As in BAEC, GRK5 overexpression induces I␬B␣ accumulation; conversely, GRK5 knockdown by GRK5siRNA associates to I␬B␣ degradation. Actin is shown for protein-loading control. HEK293 were transfected with ␬B-Luc plasmid and GRK5-WT or GRK5-siRNA and luciferase activity was measured. GRK5-WT overexpression causes inhibition of NF␬B activity, whereas GRK5 knockdown increases NF␬B transcription levels. The data in the bar graph are expressed as meanϮSEM and are representative of 3 experiments (*P Ͻ 0.05 vs. control). (E) GRK5 and I␬B␣ expression and luciferase activity were assessed in HEK293 transfected with increasing doses of GRK5siRNA.