Interferon IFN induces formation of a heterotetrameric IFN g

Interferon (IFN)-γ induces formation of a heterotetrameric, IFN-gamma-activated inhibitor of translation (GAIT) complex in human monocytic HAMI3379 (Mazumder et al., 2003a, Sampath et al., 2004). The complex binds a bipartite stem-loop element in the 3′UTR of vascular endothelial growth factor (VEGF)-A mRNA, an angiogenic factor upregulated in inflammation, and ceruloplasmin (Cp) mRNA, an acute-phase protein, and inhibits their translation (Ray and Fox, 2007, Sampath et al., 2003). The GAIT complex consists of ribosomal protein L13a, glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein-1 (NSAP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Phosphorylation of L13a has a critical role in GAIT system activation. Near-stoichiometric phosphorylation of L13a, occurring about 24 hr after IFN-γ treatment, coincides with its release from the ribosome and recruitment into the cytosolic GAIT complex (Mazumder et al., 2003a). Phosphorylated L13a (phospho-L13a) is essential for conversion of the nonfunctional pre-GAIT complex consisting of EPRS and NSAP1 to the functional, RNA-binding GAIT complex (Jia et al., 2008, Sampath et al., 2004). Moreover, phospho-L13a is the actual translation inhibitor protein; after GAIT complex binding to target mRNA, phospho-L13a interacts specifically with the eIF3-binding site of eIF4G and suppresses translation by blocking recruitment of the 43S complex (Kapasi et al., 2007). These results strongly implicate L13a phosphorylation as the rate-limiting step in GAIT-mediated translational control; however, the specific phosphorylation event(s) responsible for L13a activation and the regulatory pathway are unknown.
Results
Discussion Protein phosphorylation plays a particularly important role in translational control mechanisms. Phosphorylation of components of the translation machinery, e.g., eIF2α, eIF4B, eIF4E, eIF4G, or eEF2, is common to mechanisms regulating global translation (Dever, 2002, Raught et al., 2004), whereas phosphorylation of components of RNA-binding complexes, e.g., maskin, hnRNP K, and EPRS, is common to transcript-selective translational control (Barnard et al., 2005, Ostareck-Lederer et al., 2002, Sampath et al., 2004). We have elucidated the kinase-dependent pathway that activates GAIT-mediated translational silencing. The delayed activation of the DAPK and ZIPK cascade clarifies the molecular basis underlying the 16 hr delay before the onset of translational repression following IFN-γ treatment. The experiments reveal ribosomal protein L13a as a target of ZIPK. Importantly, the regulation of Cp and VEGF-A expression in response to IFN-γ ascribe a role to the DAPK and ZIPK cascade in the inflammatory response of myeloid cells; previous studies focused primarily on the role of these kinases in induction of apoptosis and muscle contraction (Inbal et al., 1997, Shani et al., 2004). With the addition of DAPK and ZIPK to the previously identified targets VEGF-A and Cp, it is apparent that the GAIT system constitutes a significant RNA regulon that is self limiting in nature (Keene, 2007, Keene and Tenenbaum, 2002). Negative translational regulation of the kinases inactivates the GAIT pathway and restores the cell to the basal state to allow reactivation of the IFN-γ-induced gene expression program by recurrent stimuli. This negative-feedback system defines a unique “network motif” (Milo et al., 2002) in which activation of the system is mediated by phosphorylation of an effector protein, i.e., L13a, whereas negative regulation occurs by a distinct process, namely, translational repression of the kinases (Figure 7D).
Experimental Procedures
Acknowledgments
Introduction In vertebrates, cell-specific architecture is maintained by a cytoskeleton consisting predominantly of actin filaments, microtubules, and intermediate filaments (IFs). Cytokeratins are the major structural proteins in higher eukaryotic cells (Strelkov et al., 2001, Yoon et al., 2001). Cytokeratins belong to the gene family of IFs and are specifically expressed in epithelial cells of vertebrates (Suzuki et al., 2001, Chou et al., 1997, Sagerstrom et al., 1996). IFs are composed of different, but related, structural proteins that show cell type-specific expression patterns. Thus, keratins have been utilized as useful probes to study the differentiation process of epithelial cells (Fuchs and Weber, 1994). All IF proteins share common structural motif; a central α-helical rod domain, a non-α-helical amino-terminal head domain, and a non-α-helical carboxyl-terminal tail domain. The head and tail domain often vary in length and sequence (Coulombe and Omary, 2002, Burkhard et al., 2001). A variety of keratins have been reported in vertebrates. Based on their isoelectric properties as well as molecular weight, cytokeratins are divided into two classes (Conrad et al., 1998, Druger et al., 1994). Type I consists of smaller proteins, ranging from 40 to 56 kDa, with relatively acidic isoelectric points, whereas Type II consists of larger molecules with molecular weight ranging from 53 to 67 kDa and neutral to basic isoelectric points (Imboden et al., 1997, Martorana et al., 2001). They can also be classified based on their pattern of expression; keratins expressed in epidermal keratinocytes are called E-keratins, whereas Keratins that are typical for cells forming simple epithelia are designed as S-keratins (Schaffeld et al., 1998).