JAK-STAT signaling alone is not sufficient to explain all the biological effects of IFN-y and several other kinase pathways have emerged as critical additional components of IFN-y-induced signal transduction. First of all, the phos-
phorylation of STAT1 on Y701 is not enough to induce the full expression of IFN-y-induced genes: additional phosphorylation of serine 727 is necessary (Wen et al. 1995; Kovarik et al. 2001; Varinou et al. 2003). Serine phosphorylation of STAT1 facilitates the association of chromatin-bound STAT1 with the co-activator CBP and the subsequent recruitment of histone acetylases, important for chromatin remodeling (Varinou et al. 2003). Inhibition of p38 mitogen-activated protein kinase (P38MAPK) led to defective serine phosphorylation of STAT1 in fetal brain astrocytes after stimulation with IFN-y (Lee et al. 2003). In addition, the function of serine-phosphorylated STAT1 was dependent on P38MAPK activation after stimulation of human epithelial cells or mouse fibroblasts with type I or type II IFN (Goh et al. 1999). MKK6 turned out to be the upstream activator of P38MAPK in these cells (Goh et al. 1999). Of note, dsRNA-activated protein kinase (PKR)-deficient MEFs show defective phosphorylation of S727 upon IFN-y stimulation, suggesting that PKR might function upstream of P38MAPK in these cells (Ramana et al. 2000b). In several different cell types activation of phosphatidylinositol 3-kinase (PI3K) and subsequently AKT by IFN-y are needed to phosphorylate S727 (Nguyen et al. 2001). A member of the protein kinase C (PKC) family, PKC-5, is rapidly activated in human promyelocytic cells downstream of PI3K and associates with STAT1, which then is phosphorylated on S727 (Deb et al. 2003). Notably, the activation of PKC-5 and serine phosphorylation of STAT1 are crucial for induction of pro-apoptotic genes and mitochondria-dependent apoptosis (DeVries et al. 2004). Other PKC family members might also be involved in cell type-specific responses to IFN-y. An IFN-y-induced PI3K/PKC-e/MAPK signaling pathway is involved in S727 phosphorylation in mesangial cells (Choudhury 2004). In contrast, in human embryonic kidney cells, PCK-e seems to be involved in the tyrosine phosphorylation of STAT1 (Ivaska et al. 2003), but this might occur through the activation of SRC-family kinases instead of MAPKs, as discussed below. In T cells, IFN-y activates a PI3K/mTOR/PKC-8/MKK4 signaling pathway, which does not affect the tyrosine phosphorylation of STAT1. However, since the transcription of GAS-containing genes is enhanced by this pathway, it is likely that this enhancement is also a result of increased phosphorylation of S727 (Srivastava et al. 2004). Although which MAPK is activated downstream of PI3K and PKC activation was not investigated in any of the above-mentioned studies, it is possible that a serine-threonine kinase such as P38 (Goh et al. 1999) or perhaps c-Jun kinase (JNK; Zhao et al. 2005) is directly responsible for serine phosphorylation of STAT1. The IFN-y-stimulated signaling pathway that is emerging from all of these data is: PI3K^AKT^PKC(-5, -£, or -9)^MKK(4 or 6)^P38MAPK^ serine phosphorylated STAT1. The cell type-specific variation of this proposed pathway seems to be the activation of different PKC and MKK family members.
The adaptor protein that couples the activated IFNGR to PI3K activation is presently unknown, but the CRK/CBL adaptor protein complex has been proposed to play this role (Platanias 2005).
Different cells are likely to employ alternative strategies to phosphorylate STAT1 on S727. IFN-y has been shown to elicit a calcium ion flux in thyroid cells, microglia, neutrophils, T cells, monocytes, and fibroblast-like cells, suggesting that an increase of free calcium ions is involved in IFN-y-dependent signaling in several cell types (Aas et al. 1999; Koide et al. 1988; Kung et al. 1995; Buntinx et al. 2002; Franciosi et al. 2002; Nair et al. 2002). In response to IFN-y, human fibrosarcoma cells and MEFs activate calcium/ calmodulin-dependent kinase (CAMK) II, which can interact directly with STAT1 and induce the phosphorylation of S727 in vitro (Nair et al. 2002). In keratinocytes, an increase in free calcium ions leads to activation of the annexin II/PYK2/ MEKK4/ MKK6/P38 MAPK/ATF2 signaling pathway upon IFN-y stimulation (Halfter et al. 2005). It is likely that P38 MAPK activation through this pathway also increases the serine phosphorylation of STAT1 and the consequent enhancement of ISG-transcription, although the authors did not investigate this point. Because the serine-threonine kinase CAMKII and the calcium-regulated tyrosine kinase PYK2 are both sensitive to an increase in calcium ions, it is possible that these two pathways are intertwined, particularly since it has also been described that transcription factors such as CREB, ATF, and C-EBP-ß are substrates of CAMKII (White et al. 1998; Cruzalegui et al. 2000). Indeed, IFN-y also activates CREB and C-EBP-ß in addition to ATF2 (see Sects. 6.1 and 6.2). However, more research is needed to comprehend the individual, and possibly overlapping signaling pathways that lead to the activation of these transcription factors and subsequent cell type-specific transcription. It is possible that the activation of PYK2 in certain cell types leads to downstream enhancement of the serine phosphorylation of STAT1 in addition to the activation of additional transcription factors. Interestingly, PYK2 activation by IFN-y leads to the activation of another MAPK, ERK2, eventually leading to the serine phosphorylation of STAT1 and maximal transcriptional activation in MEFs (Takaoka et al. 1999). The adaptor protein GRB2 complexed with SOS might couple the activation of PYK2 to ERK activation in response to IFN-y (Blaukat et al. 1999). However, the coupling of PYK2 with another adaptor protein such as CRK leads to activation of JNK (Blaukat et al. 1999), suggesting that IFN-y-activated PYK2 might be involved in the activation of multiple downstream signaling pathways by coupling to different adaptor proteins.
Although, as described above, IFN-y-induced PKC activation leads to MAPK activation, PKC also seems to be involved in activating SRC-family tyrosine kinases. In human alveolar epithelial cells, IFN-y activates PLC-y2 via an upstream tyrosine kinase to induce the activation of PKC-a and c-SRC or LYN, resulting in the activation of STAT1 and expression of ICAM-1, and thus the initiation of monocyte adhesion (Chang et al. 2002). SRC family kinases are required for IFN-y to activate STAT3 (but not STAT1) by tyrosine phosphorylation, whereas JAK1 and JAK2 are required to activate both STAT1 and STAT3 in MEFs (Qing and Stark 2004). FYN could be involved in STAT3 activation, because this SRC-family member associates through its SH2 domain with activated JAK2 upon IFN-y stimulation (Uddin et al. 1997). Interestingly, the tyrosine kinase PYK2 amplifies c-SRC-dependent STAT3 activation in response to epidermal growth factor (Shi et al. 2004), and it is possible that it does the same in response to IFN-y, because PYK2 becomes phosphorylated upon stimulation of MEFs by IFN-y (Takaoka et al. 1999).
In addition to affecting the activation of STATs and other transcription factors, the activation of kinases other than JAKs seems to be involved in activating other signaling pathways. For instance, the activation of mTOR downstream of PI3K leads to selective regulation of the translation of IFN-y-induced mRNAs, but not transcription, by activating p70S6K and phosphorylating the S6 ribo-somal proteins, and by phosphorylating the repressor of mRNA translation EIF4E-binding protein 1 (4EBP1), which deactivates 4EBP1, leading to its dissociation from EIF4E and the subsequent initiation of translation (Platanias 2005). In addition, treatment with IFN-y leads to the tyrosine phosphorylation of CBL, followed by the sequential activation of C3G and RAP1, resulting in subsequent growth inhibitory effects in promyelocytic cells (Alsayed 2000). Furthermore, studies performed with MEFs that lack both the a and P subunits of IKK revealed that a subset of IFN-y-induced genes is dependent on IKK activation (Sizemore et al. 2004). The IKK complex is best known as a regulator of NF-KB-dependent signaling and its effect on IFN-y-dependent signaling is currently being studied in our laboratory. Finally, IFN-y induces a MEKK1/MEK1/ERK/ C/EBP-P signaling pathway to induce the transcription of GATE-dependent ISGs (see also Sect. 6.2 and Roy et al. 2002). In summary, it is well accepted that, in addition to the JAKs, several different kinases are activated in response to IFN-y, and one can safely predict that the cell type-specific expression of these kinases and their substrates will help to determine cell type-specific responses.
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