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Phosphorylation of Glutamyl-Prolyl Trna Synthetase by Cyclin-Dependent Kinase 5 Dictates Transcript- Selective Translational Control

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Phosphorylation of Glutamyl-Prolyl Trna Synthetase by Cyclin-Dependent Kinase 5 Dictates Transcript- Selective Translational Control Phosphorylation of glutamyl-prolyl tRNA synthetase by cyclin-dependent kinase 5 dictates transcript- selective translational control Abul Arif, Jie Jia, Robyn A. Moodt, Paul E. DiCorleto, and Paul L. Fox1 Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 Edited by Paul Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved December 9, 2010 (received for review July 30, 2010) Cyclin-dependent kinase 5 (Cdk5) is an atypical but essential (ERS) and prolyl tRNA synthetase (PRS) domains joined by a member of the Cdk kinase family, and its dysregulation or deletion noncatalytic linker of three highly structured WHEP-TRS repeats has been implicated in inflammation-related disorders by an separated by putative unstructured spacers (Fig. 1A). The linker undefined mechanism. Here we show that Cdk5 is an indispens- by itself is sufficient to bind the other GAIT components and re- able activator of the GAIT (IFN-γ–activated inhibitor of translation) constitute a functional GAIT complex in vitro (9, 13). pathway, which suppresses expression of a posttranscriptional IFN-γ induces early release of EPRS from the MSC and con- regulon of proinflammatory genes in myeloid cells. Through in- sequent binding to NSAP1 to form the pre-GAIT complex, which duction of its regulatory protein, Cdk5R1 (p35), IFN-γ activates lacks GAIT element RNA-binding activity. After ∼14 h, phos- Cdk5 to phosphorylate Ser886 in the linker domain of glutamyl- phorylated L13a and GAPDH interact with the pre-GAIT com- prolyl tRNA synthetase (EPRS), the initial event in assembly of plex to form the four-protein GAIT complex that binds and inhibits the GAIT complex. Cdk5/p35 also induces, albeit indirectly via a dis- translation of GAIT element–bearing RNA. These events are tinct kinase, phosphorylation of Ser999, the second essential event mediated by IFN-γ–inducible, two-site phosphorylation of EPRS in GAIT pathway activation. Diphosphorylated EPRS is released in the linker domain at Ser886 and Ser999 after ∼1 and 2 h, re- from its residence in the tRNA multisynthetase complex for imme- spectively (9). Both phosphorylation events are required for the diate binding to NS1-associated protein and subsequent binding release of EPRS from the parent MSC. Negative regulation by to ribosomal protein L13a and GAPDH. The mature heterotetra- NSAP1 specifically requires phosphorylation at Ser886. Ser999 meric GAIT complex binds the 3′ UTR GAIT element of VEGF-A phosphorylation is essential for conformationally correct in- and other target mRNAs and suppresses their translation in mye- teraction with phospho-L13a and GAPDH to generate the active loid cells. Inhibition of Cdk5/p35 inhibits both EPRS phosphoryla- GAIT complex that binds target RNA and interacts with eIF4G of tion events, prevents EPRS release from the tRNA multisynthetase the translation initiation machinery, thereby inhibiting translation. complex, and blocks translational suppression of GAIT element– Significant advances have been made in understanding the role bearing mRNAs, resulting in increased expression of inflammatory of phosphorylation in inducing noncanonical functions of AARSs. proteins. Our study reveals a unique role of Cdk5/p35 in activation Information on the upstream signaling events and the proximal of the major noncanonical function of EPRS, namely translational kinases that induce these phosphorylation events in cells is lim- control of macrophage inflammatory gene expression. ited, however. Here we report an IFN-γ–induced signaling event that activates cyclin-dependent kinase (Cdk)-5 and its regulatory minoacyl-tRNA synthetases (AARSs) are ubiquitous en- protein, Cdk5R1 (p35), which directly catalyzes the initial EPRS Azymes with evolutionarily conserved catalytic cores that per- Ser886 phosphorylation event and indirectly induces Ser999 phos- form their major function, ligation of amino acids to cognate phorylation through activation of a distinct Ser999 kinase. Cdk5/ tRNAs for protein synthesis (1, 2). AARSs also contribute to key p35-mediated induction of EPRS phosphorylation and GAIT biological processes beyond their canonical activities (3, 4). In most system activation might be an important link between kinase and cases, AARS phosphorylation initiates the noncanonical function macrophage inflammatory gene expression. with little disruption of aminoacylation activity (5). Phosphoryla- tion of ThrRS, SerRS, and LysRS increases the synthesis of Ap4A, Results a pleiotropic signaling molecule (6, 7). In myeloid cells, IFN-γ– Cdk5 Phosphorylates EPRS at Ser886. Previous pharmacologic in- induced phosphorylation of glutamyl-prolyl tRNA synthetase hibitor studies have suggested that an unidentified proline- (EPRS) initiates transcript-selective translational inhibition of in- directed kinase is responsible for EPRS Ser886 phosphorylation flammatory mRNAs (8, 9). Stimulus-dependent phosphorylation (9) (Fig. 1A). Site-specific mutagenesis demonstrated the re- of EPRS drives the release of EPRS from its residence in the tRNA quirement for S/T-P-X-K/R, a proline-directed kinase recognition multisynthetase complex (MSC) to facilitate the noncanonical motif, in the linker sequence 886SPTR. Inhibitor studies also activity (8, 9). Stimulus- and phosphorylation-dependent release of showed that a distinct AGC kinase phosphorylates EPRS at Ser999 other MSC components has been reported, supporting the “depot (9). To identify the specific Ser886 kinase, candidate proline- hypothesis” in which proteins are inducibly released from macro- directed kinases were subjected to immunoprecipitation using molecular complexes to perform auxiliary, “moonlighting” func- antibodies against multiple MAPKs and Cdks. Site-specific tions (7, 10). These previous studies showed that signal-dependent phosphorylation of Ser886 was measured by in vitro phosphoryla- phosphorylation is critical to the regulation of AARS conforma- CELL BIOLOGY tion, activity, intracellular localization, protein (and RNA) part- ners, and, ultimately, noncanonical function. Author contributions: A.A., J.J., P.E.D., and P.L.F. designed research; A.A., J.J., and R.A.M. EPRS is a component of the GAIT complex that binds the performed research; A.A., J.J., and P.L.F. analyzed data; and A.A., P.E.D., and P.L.F. wrote 3′ UTR GAIT element in multiple proinflammatory transcripts the paper. (e.g., VEGF-A) and inhibits their translation in human myeloid The authors declare no conflict of interest. cells (11, 12). The GAIT complex comprises EPRS, NS1-associated This article is a PNAS Direct Submission. protein (NSAP1), ribosomal protein L13a, and GAPDH (8). A 1To whom correspondence should be addressed. E-mail: [email protected]. unique GAIT component that recognizes and directly binds target This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mRNAs, EPRS consists of distinct glutamyl tRNA synthetase 1073/pnas.1011275108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011275108 PNAS | January 25, 2011 | vol. 108 | no. 4 | 1415–1420 Downloaded by guest on September 25, 2021 γ t. experiments with pharmacologic inhibitors (9). The roles of A IFN- D n siRNA: Cont. ERK2 Cont. Cdk1 Co Cdk5 ERK2 and Cdk5 in EPRS phosphorylation were verified by Kinase Proline-directed kinase probing endogenous protein with phosphospecific EPRS anti- P- IB, α-ERK2 IB, α-Cdk1 IB, α-Cdk5 EPRS bodies. siRNA targeting ERK2 and Cdk5, but not Cdk1, signifi- Pi AGC kinase Ser886 886 999 Pi IB, α-P-EPRS-Ser886 cantly inhibited both Ser and Ser phosphorylation (Fig. 1D). P- ERS PRS EPRS Identical results were obtained using primary human peripheral 999 Ser α 999 P P IB, -P-EPRS-Ser blood monocytes (PBMs) (Fig. S1). 886 999 886 Ser Ser Actin To identify the proximal Ser kinase, recombinant ERK2 and IB, α-Actin Cdk5 were allowed to in vitro phosphorylate EPRS linker mu- tated to permit phosphorylation of Ser886 or Ser999. Cdk5 pre- IFN-γ ERK2: B E Cdk5/p35: activated with p35 activator protein phosphorylated EPRS at 2 3 te e K 886 at ERK1 S999A linker: Ser , suggesting that Cdk5 is the proximal kinase (Fig. 1E). ys -Cdk -GS IP Ab.: Lysa L α- α-ERK2α-Cdk1α α-Cdk5α S886A linker: Cdk5 did not phosphorylate Ser999, consistent with its role as an P-GST upstream activator of a distinct kinase. ERK2 did not phos- P-Linker p35 Ser886 P-EPRS phorylate either site, indicating that it is upstream of Cdk5. The Immunocomplex kinase assay linker phosphorylation of p35 by Cdk5 has been reported previously In vitro phos., [γ-32P]ATP (14). To show that cellular Cdk5 phosphorylates Ser886, the ki- nase was immunoprecipitated from IFN-γ–activated U937 cell C F up. IP siRNA: Control ERK2 Cdk5 Cdk1 Input S lysates and used to in vitro phosphorylate the linker target. The P-Ser886 pcDNA3- A A A 9A 6 9A Linker immunoprecipitate and supernatant phosphorylated histone H1, Flag- 99 8 99 99 linker: WT S886AS WT S886AS9 WT S886AS999WT S8 S In vitro phos. linker S999A a general Cdk substrate (15) (Fig. 1F); however, only Cdk5-im- P- P-Histone 886 Linker H1 munoprecipitate phosphorylated Ser -containing linker, to an 32 α P-labeling, IP, -Flag In vitro phos., histone H1 extent comparable to the total activity in IFN-γ–activated cell Linker Cdk5 lysate. These results provide compelling evidence that Cdk5 is the IB, α-Flag IB, α-Cdk5 proximal kinase responsible for IFN-γ–stimulated EPRS phos- 886 Fig. 1. ERK2 and Cdk5 are required for phosphorylation of EPRS at Ser886. phorylation at Ser . (A) Schematic of the IFN-γ–activated kinase pathway directing
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