Regulation by Posttranslational Modifications

In quiescent cells, STAT proteins exist as inactive monomers. One of the key signals that triggers the activation of STATs is through tyrosine phosphorylation. A single conserved tyrosine residue, which is located immediately after the SH2 domain of STAT proteins, becomes rapidly phosphorylated after ligand stimulation. For example, IFN treatment of cells triggers the phosphorylation of Tyr701 residue of STAT1 (Shuai et al., 1993a). Mutation of Tyr701 to Phe abolishes the dimerization, DNA binding, and nuclear translocation of STAT1 (Shuai et al, 1993a). Thus, tyrosine phosphorylation plays a critical role in the activation of STATs.

In addition to tyrosine phosphorylation, STAT1, STAT3, STAT4, STAT5A, and STAT5B have also been shown to be modified by phosphorylation at a serine residue located in the COOH-terminal transcriptional activation domain (Decker and Kovarik, 2000). Serine phosphorylation and tyrosine phosphorylation of STATs are independent events. STATs are constitutively serine phosphorylated, which can be further enhanced by cytokine stimulation. Mutational analysis indicates that serine phosphorylation is required for the maximum transcriptional activity of STATs (Wen and Darnell, 1997; Wen et al, 1995). Several serine kinases, including extracellular signal-regulated protein kinase (ERK), p38, JUN N-terminal kinase (JNK), and protein kinase C8 (PKC8) have been suggested to participate in STAT serine phosphorylation under different conditions (Decker and Kovarik, 2000). It has been suggested that serine phosphorylation of STAT1 is required for the interaction of STAT1 with the co-activator CREB-binding protein (CBP), a histone acetyltransferase (Varinou et al, 2003).

Recently, the physiological importance of STAT serine phosphorylation has been investigated. The STAT1S727A mutant mice, in which the serine phosphorylation site of STAT 1 (Ser727) was substituted with alanine, had been generated (Varinou et al, 2003). The transcription of only a subset of IFN-y-responsive genes was reduced in STAT1S727A macrophages. Furthermore, when infected with lower doses of bacteria, the StatlS727A and wild-type control mice exhibited similar survival rates. However, the mutant STAT1S727A mice displayed increased mortality when challenged with higher doses of bacteria (Varinou et al, 2003). Thus, these studies demonstrate that serine phosphorylation of STAT1 plays a critical role in IFN-mediated innate immunity to highly pathogenic infection, but serine phosphorylation of STAT1 is dispensable for host defense to a milder pathogenic infection.

The physiological importance to STAT3 serine phosphorylation has also been examined by gene targeting studies. Mice carrying a mutant STAT3 gene with the serine727 residue substituted to alanine (STAT3S727A) have been created (Shen et al., 2004). The transcriptional activation of STAT3-dependent genes in response to IL-6 and oncostatin M (OSM) was significantly reduced in embryonic fibroblasts derived from the STAT3S727A mice (SA/SA). Surprisingly, in contrast to the STAT3-null (-/-) mice which were embryonic lethal, the SA/SA mice showed no defect in development. However, STAT3 SA/null (SA/-) mice displayed perinatal lethality and growth retardation (Shen et al., 2004), while STAT3 wild type/null (+/-) were normal in animal development. These results support the importance of STAT3 serine phosphorylation under certain physiological conditions.

STATs can be modified by protein acetylation. Recently, it has been reported that STAT3 becomes acetylated on a single lysine residue, Lys685, in response to cytokine stimulation (Yuan et al., 2005). The histone acetyltransferase p300 catalyzes the acetylation of Lys685, which can be removed by type I histone deacetylase (HDAC). Mutational analysis suggests that Lys685 acetylation is important for the formation of STAT3 dimer as well as the DNA binding and transcriptional activity of STAT3 (Yuan et al, 2005).

Protein methylation has been suggested to regulate the activity of STATs (Mowen et al, 2001). STAT1 was reported to be methylated on Arg31 by protein arginine methyl-transferase I (PRMT1). It was proposed that arginine methylation of STAT1 increases the DNA binding activity of STAT1 (Mowen et al., 2001). However, other studies have also been reported that argue against the possible modification of STAT1 by protein methylation (Meissner et al, 2004). Thus, the role of protein methylation in the regulation of STAT activity remains to be clarified.

The ubiquitin-proteasome pathway, which targets protein for degradation in various fundamental cellular processes such as transcriptional control, apoptosis, and cell cycle regulation (Pickart, 2001), has been suggested to regulate STAT signaling. For example, polyubiquitination of STAT1 has been reported (Kim and Maniatis, 1996). However, the newly synthesized STAT1 protein was found to be rather stable by pulse-chase analysis (Haspel et al, 1996). Thus, the physiological importance of protein ubiquitination in the regulation of STAT protein stability is still unclear.

Protein sumoylation has recently been implicated in the regulation of STAT signaling. SUMO conjugation occurs through a pathway that is distinct from, but analogous to, protein ubiquitination (Johnson, 2004). Protein sumoylation has been suggested to regulate a wide variety of biological processes, including protein stability, protein-protein interaction, protein localization, and modulation of transcription factors (Johnson, 2004). The Lys703 residue of STAT1 was found to be sumoylated (Rogers et al., 2003; Ungureanu et al., 2003). As will be discussed in the next section, PIAS proteins, which possess SUMO E3 ligase activity, have been shown to promote STAT1 sumoylation. However, contradictory results have been reported on the role of Lys703 sumoylation in the regulation of STAT 1 activity (Rogers et al., 2003; Ungureanu et al., 2005; Ungureanu et al., 2003). Further studies are needed to clarify the physiological role of protein sumoylation in STAT signaling.

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