Tatsuji Matsuoka

Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis

Protein Ser/Thr kinase CK2 (casein kinase II) is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-GlcNAc on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344) on the same protein. We use protein semisynthesis to show that Thr344 phosphorylation increases CK2α cellular stability via Pin1 interaction whereas Ser347 glycosylation appears to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with the site-specifically modified phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that CK2 kinase substrate selectivity is modulated by these specific posttranslational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

Identification of 2′-Phosphodiesterase, Which Plays a Role in the 2-5A System Regulated by Interferon

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5′-triphosphorylated, 2′,5′-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2′,5′-oligoadenylate synthetases (2′,5′-OASs), inactivated by 5′-phosphatase and completely degraded by 2′-phosphodiesterase (2′-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2′,5′-OASs and RNase L have been molecularly cloned and studied well, the identification of 2′-PDE has remained elusive. Here, we describe the first identification of 2′-PDE, the third key enzyme of the 2-5A system. We found a putative 2′-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2′-PDE by mass spectrometry. Based on the full-length sequence of the human 2′-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2′-PDE expressed in mammalian cells certainly cleaved the 2′,5′-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2′-PDE were found, the human 2′-PDE was considered to be a unique enzyme without isoform. Suppression of 2′-PDE by a small interfering RNA and a 2′-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2′-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2′-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

Purification and Identification of Activating Enzymes of CS-0777, a Selective Sphingosine 1-Phosphate Receptor 1 Modulator, in Erythrocytes

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ∼10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.