Reversible protein phosphorylation is an essential regulatory mechanism in many cellular processes. Cells use this post-translational modification to alter the activity or localization of key regulatory proteins. Tyrosine and serine/threonine protein phosphatases are highly abundant proteins present in many cellular compartments in mammalian cells. Together with kinases, they set the phosphorylation state of signaling and effector proteins and thereby play a large role in controlling cellular responses. Inappropriate or defective phosphatase or kinase activity leads to aberrant patterns of phosphorylation. Dramatic changes in phosphorylation of many proteins were demonstrated during global ischemia, including enriched phosphatase activity in reactive astrocytes (Hasegawa et al., 2000
). To date there has not been a systematic examination of phosphatase activity in astrocytes.
Here we report a large-scale classification of phosphatases focused on their control of NF-κB-mediated transcriptional activity. Nineteen phosphatases were identified to participate in either up- or down-regulation of NF-κB activity in astrocytes. Most of these phosphatases were not previously known to associate with this pathway. The involvement of additional phosphatases can not be excluded as rigid criteria and a high threshold of NF-κB activity were used to identify candidate genes. Stimulus and cell specificity, compensatory or redundant pathways, and the presence of non-functional siRNAs may cause additional underestimates of the number of phosphatase genes involved in NF-κB transcriptional activity.
At least 13 phosphatases were previously implicated in NF-κB signaling, including PPP2CA (Yang et al., 2001
), PPM1B (Prajapati et al., 2004
), PPM1L (Li et al., 2003
; Takaesu et al., 2003
), INPP4A (Franke et al., 1997
; Romashkova and Makarov, 1999
), PTEN (Mayo et al., 2002
), PTPN2 (Ibarra-Sanchez et al., 2001
), PPP4C (Hu et al., 1998
), CDC25B (Zheng et al., 2004
), PPP6C (Bouwmeester et al., 2004
), PPP2R1A (Zheng et al., 2004
), PPP2R1B (Zheng et al., 2004
), PPP2R5C (Moreno et al., 2004
), and DUSP5 (Zheng et al., 2004
). Nine of these genes were also identified by the present analysis although the mechanisms by which most of these phosphatase genes impact NF-κB signaling are poorly understood. The four genes missed in our screen include DUSP5, however, the murine homolog of DUSP5 has not been identified. Silencing CDC25 phosphatases, which are critical to mitotic entry, markedly inhibited Renilla
luciferase activity suggesting damage to the target cells; therefore, analysis of CDC25B was not pursued. RNAi to PPP6C inhibited basal NF-κB reporter activity but failed to meet the threshold established for our screening. PPM1B (also termed PP2Cβ) bound and dephosphorylated IKK in human HeLa and 293 embryonic kidney cells. However, we failed to detect any activity of PPM1B on NF-κB activity in mouse astrocytes (), even though the RNAi constructs effectively inhibited mRNA levels (Supplementary Fig. 2Z
) and modulated NF-κB reporter activity in mouse fibroblasts (). Reciprocally, 3 phosphatases that regulated NF-κB activity in astrocytes failed to modulate NF-κB reporter activity in fibroblasts. These results suggest potential cell type specificity in the activity of phosphatases on NF-κB signaling, an observation with potential implications for controlling inflammation in various clinical conditions.
PP2A enzymes regulate at least three different steps in the NF-κB pathway including TRAF2, IKK, and NF-κB p65 (). Previous studies showed that the activity of IKK on IκB kinase was associated with PP2A and down regulated by the PP2A catalytic subunit (DiDonato et al., 1997
; Fu et al., 2003
). We observed selective non-redundant utilization of specific catalytic and structural chains in the core enzyme complexes, i.e. PPP2CB/PPP2R1A were selectively coupled to the IKK complex while PPP2CA/ PPP2R1B were physically and functionally associated with the p65 NF-κB complex (). Although the PP2A complex was shown to bind and dephosphorylate the p65 chain of NF-κB (Yang et al., 2001
), there was no description of the composition of the PP2A enzyme. The present report functionally extends these observations by identifying PPP2CA and PPP2R1B as the NF-κB interactive chains () and demonstrates the selective dephosphorylation of the Ser536
residue in the NF-κB p65 subunit. Our data suggest the potential of multiple corresponding site specific-phosphatases for NF-κB p65.
In addition, we identified a PP2A holoenzyme associated with TRAF2 (). Analysis of this interaction demonstrated Thr117 in the first TRAF2 zinc finger domain is a phosphorylation site and phosphorylation of Thr117 is required for TRAF2-mediated-NF-κB activity (). The present data also demonstrate ligand-induced phosphorylation of TRAF2 and suggest TRAF2 may be the target of the PP2A holoenzyme (). Future experiments will address the mechanisms involved in TRAF2 phosphorylation.
The PP2A chains combine in different combinations to form core enzymes and holoenzymes. In mice the PPP2CA and PPP2CB catalytic chains are 97% identical and the structural chains are 86% identical. However, PPP2CA null mutant mice were embryonic lethal, demonstrating that PPP2CA is an essential non-redundant gene (Gotz et al., 1998
). In the present study co-immunoprecipitation showed non-redundant PP2A catalytic and structural chains were preferentially associated with their substrate. This suggests selective combinations of non-redundant PP2A catalytic and structural chains may be critical for substrate targeting.
Several phosphatases regulated basal NF-κB activity suggesting that NF-κB activity is tightly regulated and may be required for cellular homeostasis. Indeed, phosphorylation of p65 and its shuttling in and out of the nucleus have been observed in several cell types including astrocytes (Zhai et al., 2004
). Basal NF-κB activity was reported to be critical for protecting cells from apoptosis (Bureau et al., 2002
). Constitutive NF-κB activity has also been detected in glioblastomas and other tumors. The molecular mechanisms responsible for altered regulation of the NF-κB pathway in cancer cells remain largely unknown but some phosphatase genes (eg. PTPRJ and PPP2CB) identified in this report sensitize or promote cell death (MacKeigan et al., 2005
) and therefore hold potential roles as tumor suppressors.
Astrocytes are implicated in the pathophysiology of neurodegenerative and inflammatory diseases including Alzheimer's disease and multiple sclerosis (Miller, 2005
). These diseases are characterized by scarring lesions containing reactive hypertrophic astrocytes. These reactive astrocytes are a major source of chemokines that orchestrate migration and activation of leukocytes and microglial cells into neuronal lesions. The knowledge that phosphatases identified in this report can selectively regulate chemokine and cytokine expression (Fig. 6) offers new therapeutic targets with the potential of regulating inflammatory diseases.