Stimulation of NFκB Activity by Multiple Signaling Pathways Requires PAK1

Abstract

The p21-activated kinase (PAK1) is a serine-threonine protein kinase that is activated by binding to the Rho family small G proteins Rac and Cdc42hs. Both Rac and Cdc42hs have been shown to regulate the activity of the transcription factor NFB. Here we show that expression of active Ras, Raf-1, or Rac1 in fibroblasts stimulates NFB in a PAK1-dependent manner and that expression of active PAK1 can stimulate NFB on its own. Similarly, in macrophages activation of NFB as well as transcription from the tumor necrosis factor ␣ promoter depends on PAK1. In these cells lipopolysaccharide is a potent activator of PAK1 kinase activity. We also demonstrate that expression of active PAK1 stimulates the nuclear translocation of the p65 subunit of NFB but does not activate the inhibitor of B kinases ␣ or ␤. These data demonstrate that PAK1 is a crucial signaling molecule involved in NFB activation by multiple stimuli. NFB1 is a transcription factor that is critically involved in cellular growth and transformation, the suppression of apoptosis, and the response to inflammatory stimuli (1-6). It consists of homo-and heterodimers of members of the Rel family of transcription factors (1, 2). The most frequently studied form of NFB consists of two proteins, p50 (NFB1) and p65 (RelA). In unstimulated cells, this heterodimer is retained in the cytoplasm by an inhibitory protein known as the inhibitor of B (IB). In response to most stimuli that activate NFB, IB becomes phosphorylated, ubiquitinated, and subsequently degraded by the 26 S proteosome (7,8). Once free of IB, NFB translocates to the nucleus and activates the transcription of target genes. The transcriptional activity of NFB is also controlled by phosphorylation of dimer subunits. For example, both p50 and p65 are phosphorylated in cells, and phosphorylation of p65 has been shown to positively regulate its transcriptional activity (9 -14).The phosphorylation of IB that leads to its degradation occurs on two conserved serine residues within its N terminus (serines 32 and 36 in IB␣) (15). Two kinases have been identified and cloned that will phosphorylate both of these sites in cells, namely IB kinase ␣ and IB kinase ␤ (IKK␣ and IKK␤, respectively) (16 -20). Based on knockout studies in mice, IKK␤ seems to be more important than IKK␣ for controlling NFB activity in response to cytokines and other ligands (21-25). Both of these kinases are present in a high molecular weight protein complex that contains at least two distinct scaffolding proteins (IKK␥ and IKK complex-associated protein), as well as NFB, IB, and other proteins (26 -29). The activities of IKK␣ and IKK␤ are controlled by the related kinases NFB-interacting kinase (NIK) and the mitogen-activated protein/extracellular signal-regulated kinase kinase kinases 1-3 (MEKK1-3) (30 -36). Other kinases that control IKK activity may yet be identified. Recent studies have shown that two upstream regulators of NIK are the transforming growth factor-␤-activated kinase 1 and Cot/Tpl2, whereas MEKK1 has been shown to be regulat...

Ral GTPases Contribute to Regulation of Cyclin D1 through Activation of NF-κB

“…Either of these activities conceivably couples to regulation of NF-B and cyclin D1. For example, Rac can regulate NF-B and cyclin D1 induction through activation of p21-activated protein kinase and other downstream effectors (17,24,46). In contrast to negative regulation of Rac, sequestration of RalBP1 by Ral-GTP possibly elevates basal levels of active Rac.…”

“…pCH110 constitutively expresses the lacZ gene from a simian virus 40 promoter (Pharmacia Biotech, Inc.). pDCR-ras12V, pDCR-ras12V,37G, pRSV-p65, pCMV-IKK␤KM, pCEP4-I B␣ SS/AA, pcDNA3-HA-NIK, pcDNA3myc-NIK (EE429/430AA), and pEGFP-p65 are described elsewhere (2,17,52,54,59).…”

“…The coding sequence for ralB23V was inserted as a BamHI fragment into pBabePuro to create pBabePuro-ralB23V. Luciferase reporter plasmids 2X NF-B-Luc, -1745 CD1-Luc, -66 CD1-Luc, -66 CD1-Bmut-Luc, and -66 ATFmut1-Luc are described elsewhere (17,21). To create -66 ATFmut2-Luc, the region of -66 CD1-Luc containing the activating transcription factor (ATF) binding site consensus sequence was mutated from 5Ј-TAACGTCACACGGACT-3Ј to 5Ј-T cgCGTCAccCGGACT-3Ј (mutated bases are in lowercase) by PCR using human cyclin D1-specific primers.…”

Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKKε

“…Other groups have observed RelA/p65 nuclear translocation independent of the classical IKK-I B pathway: coexpression of PAK1 was shown to induce RelA/p65 nuclear translocation independent of IKK␣/IKK␤ activity or I B␣ Ser 32 /Ser 36 phosphorylation (53). Recently, p53 was shown to induce NF-B activation through RSK-1-mediated phosphorylation of RelA/p65 in an I B␣ degradation-independent manner (54).…”

Rac1 Negatively Regulates Lipopolysaccharide-Induced IL-23 p19 Expression in Human Macrophages and Dendritic Cells and NF-κB p65 trans Activation Plays a Novel Role

“…Although Rac1 can activate NF-B in various types of cells (17)(18)(19), the role of Rac1 in NF-B activation and cytokine expression in human macrophages and DCs has not been determined.…”