An obligatory step in the activation of Signal Transducers and Activators of Transcription (STATs) by cytokines is their docking to specific receptors via phosphotyrosines. However, this model does not address whether STATs pre-associate with their corresponding receptor or exist free in the cytoplasm before receptor activation. In this report, we demonstrate that pre-association of STAT1 with the receptor is required for type I interferon (IFN) signaling. Interestingly, the interaction between the human type I IFN receptor and STAT1 is not direct but mediated by the adapter protein receptor for activated protein kinase C (RACK1). Disruption of the IFN␣ receptor-RACK1 interaction abolishes not only IFN␣-induced tyrosine phosphorylation of STAT1 but also activation of STAT2, indicating that RACK1 plays a central role in early signaling through the Jak-STAT pathway. These findings demonstrate the involvement of RACK1 in STAT1 activation and raise the possibility that other STATs may pre-associate with cytokine receptors through similar adapter-STAT-mediated interactions. Cytokines and interferons (IFNs)1 bind to receptors of the cytokine receptor superfamily (1-3), resulting in the activation of kinases of the Jak family and transcription factors designated STATs or Signal Transducers and Activators of Transcription (4 -7). The Jak-STAT pathway has evolved as the paradigm of cytokine and IFN signaling (4 -7). Although STAT can be activated by different cytokines (i.e. STAT1 is activated by IFN␣, IFN␥, IL6, leukemia inhibitory factor, IL10, etc.), studies with knockout mice clearly indicated that their function is well restricted to precise systems. For example, STAT1 is only required for the physiological functions of IFN␣ and IFN␥ (reviewed in Ref. 7).STATs are recruited to distinct phosphotyrosines within the receptor complex and then are phosphorylated, probably by Jaks, on the highly conserved C-terminal tyrosines (i.e. tyrosine 701 of Stat1), allowing the SH2 domain of one STAT to interact with the phosphorylated tyrosine on another STAT to form homo-or heterodimers. STAT dimers translocate to the nucleus, where they bind specific DNA elements to activate or inhibit transcription of specific genes (reviewed in Refs. 5 and 8).One distinctive feature in the type I IFN system is that STAT2 is pre-associated with IFN␣R␤L chain (9, 10). Activation of STAT2 in response to type I IFNs (IFN␣, ␤, or ) requires the presence of this constitutive site and one or more of the five proximal tyrosines of the ␤L chain (9). However, the mechanism for STAT1 activation by type I IFNs has not been elucidated. It is known that activation of STAT1 requires the previous activation of STAT2 (11), but it has not been determined whether receptor tyrosines are also required for activation. This is in clear contrast to the activation of STAT1 by IFN␥, which requires docking of STAT1 to a phosphorylated tyrosine on the ␣ chain of the receptor (12).We have recently reported (13) that RACK1, originally described as a Receptor for Activated ...

Kinases of the Jak family (Jak1/2/3 and Tyk2) interact with the membrane proximal domain of different cytokine receptors and play a critical role in the activation of cytokine and growth factor signaling pathways. In this report we demonstrate that both the Box 1 and Box 2 motif collaborate in the association and activation of Jak1 by type I interferons. Mutational analysis of the ␤ chain of type I interferon receptor (IFN␣R␤L/IFNAR2) revealed that Box 1 plays a more significant role in activation than in the association with Jak1. On the contrary, the Box 2 motif contributes more to the association with Jak1 than to kinase activation. Additionally, the study of the Jak1 binding sites on the IL2 receptor ␤ (IL2R␤), IFN␥R␣/IFNGR1, and IL10R␣/IL10R1 chains suggests that cytokine receptors have two different kinds of interaction with Jak1. One form of interaction involves the Box 1 and the previously described Box 2 motif, which we now designate as Box 2A, characterized by the VEVI and LEVL sequences present in IFN␣R␤L/IFNAR2 and IL2R␤ subunits, respectively. The second form of interaction requires a motif termed Box 2B, which is present in the IFN␥R␣/IFNGR1 (SILLPKS) and IL10R␣/IL10R1 (SVLLFKK) chains. Interestingly, Box 2B localizes close to the membrane region (8 -10 amino acids from the membrane) similar to Box 1, whereas Box 2A is more distal (38 -58 amino acids from the membrane).

The human type I interferon (IFN) 1 family is composed of multiple subtypes of IFN␣ (IFN␣1, IFN␣2, etc.), IFN␤, and IFN (1). The human type I IFN receptor (IFN-R) or IFN␣␤R is composed of at least two subunits, termed ␣ and ␤ (also designated as IFNAR1 and IFNAR2, respectively). The ␤ subunit has two transmembrane forms ␤ Short (␤ S ) and ␤ Long (␤ L ), both of which can bind IFNs with low affinity (2). High affinity binding occurs when both ␣ and either form of ␤ are coexpressed; however, in the absence of ␤, the ␣ chain cannot bind IFN (reviewed in Ref.3). Each chain also associates with a specific Jak kinase that is required for signaling; the ␣ chain docks Tyk2 (4, 5), whereas the ␤ L subunit interacts with Jak1 (6). Oligomerization of the receptor subunits occurs upon ligand binding and induces intra-and intermolecular phosphorylation of the Jak kinases (reviewed in Refs. 7-10). The activated kinases are then thought to phosphorylate the receptor chains and Stats 1, 2, and 3. The Stat molecules then homo-or heterodimerize and migrate to the nucleus where they stimulate transcription of the IFN-inducible genes (10 -13).In most cytokine systems tyrosine phosphorylation of the receptor subunits was originally thought to be required for recruitment and phosphorylation of the Stat factors by the receptor-Jak kinase complex. This model holds true for some cytokine systems such as the IFN␥ and interleukin 6 systems, where specific tyrosines in the ␣ and gp130 subunits were shown to be essential for recruitment and phosphorylation of Stat1 and Stat3, respectively (14,15). However, recent reports indicate that not all cytokine systems require receptor phosphorylation for Stat activation, i.e. growth hormone and granulocyte-colony-stimulating growth factor receptors devoid of tyrosines can still activate Stat proteins (16 -18).In the type I IFN system, the role of tyrosine phosphorylation of the type I IFN-R in Stat binding and activation is not clear (19 -21). For example, certain cell lines expressing a variant form of the type I receptor, which fails to phosphorylate the ␣ subunit, are still capable of producing an antiviral and antiproliferative effect in response to IFN␣2 (22). Moreover, Gibbs et al. (23) showed that expression of a human ␣ chain devoid of tyrosines in mouse L-929 cells did not impair formation of the Stat1-and Stat2-containing ISGF3 complex. However, it is worth mentioning that the role of the ␤ chain of the receptor in Stat activation has not been explored.This report seeks to answer several related questions regarding the mechanisms of Stat activation by type I IFNs. First, what receptor domains are required for Stats 1 and 2 activation? Second, what is the importance of tyrosine phosphoryla-

The interaction between receptors and kinases of the Janus kinase (Jak) family is critical for signaling by growth factors, cytokines, and IFNs. Therefore, the characterization of the domains involved in these interactions is pivotal not only in understanding kinase activation but also in the development of drugs that mimic or inhibit signaling. In this report, we have characterized the domains of Jak1 required to associate with distinct cytokine receptor subunits: IFN-αRβL, IFN-γRα, IL-10Rα, IL-2Rβ, and IL-4Rα. We demonstrate that two regions of Jak1 are necessary for the interaction with cytokine receptors. First, a common N-terminal region that includes Jak homology (JH) domain 7 and the first 19 aa of JH6, and, second, a C-terminal region (JH6–3) that was different for distinct receptors. The contribution of the two different regions of Jak1 to cytokine receptor binding was also variable. Deletion of JH7–6 impaired the association of IL-2Rβ and IL-4Rα chains with Jak1 but did not have a major impact on the binding of Jak1 to IFN-αRβL or IL-10Rα. Interestingly, regardless of the effect on receptor binding, removal of JH7–6 completely abrogated kinase activation, indicating that this domain is required for ligand-driven kinase activation and, thus, for proper signaling through cytokine receptors.

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-αRβL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-αRβL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-αRβL, the minimum site for RACK-1 binding was mapped to aa 300–346. RACK-1 binding to IFN-αRβL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-αRβL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-αRα), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-αRβL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-β1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-β1b. However, stimulation of cells with either IFN-β1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.

No abstract

No abstract

The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-α receptor 2/β subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that includes the IFN-α receptor 2/β-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin.