Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

Abstract

MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position ؊3 (P؊3) being more stringent than for prolines at P؊2 and P؉1. Using the consensus, we identified a putative phosphorylation site (RPLT 1080 S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.

Intestinal Cell Kinase (ICK) Promotes Activation of mTOR Complex 1 (mTORC1) through Phosphorylation of Raptor Thr-908

“…9. Briefly, cells were harvested in ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 2 mM EGTA, and supplemented with complete protease inhibitors (Roche), 1 mM Na 3 VO 4 , 1 M microcystin LR, and 5 mM ␤-glycerophosphate).…”

“…Unlike MAPKs, the ICK kinase activity cannot be acutely stimulated by serum or growth factors. Our prior studies suggest that protein phosphatase 5 (7) and CCRK/CDK20 (cell cycle-related kinase/cyclin-dependent kinase 20) (8) are putative upstream yin-yang regulators for the phosphorylation of ICK Thr-157 (9). We have shown that oxidative stress, which activates protein phosphatase 5, is able to inactivate ICK by dephosphorylating Thr-157 within the TDY motif (9).…”

“…Our prior studies suggest that protein phosphatase 5 (7) and CCRK/CDK20 (cell cycle-related kinase/cyclin-dependent kinase 20) (8) are putative upstream yin-yang regulators for the phosphorylation of ICK Thr-157 (9). We have shown that oxidative stress, which activates protein phosphatase 5, is able to inactivate ICK by dephosphorylating Thr-157 within the TDY motif (9). Although CCRK is closely related to yeast Cak1p (Cdk-activating kinase) and mammalian CDK7 (cyclin-dependent kinase 7), there is controversy as to whether CCRK has any intrinsic CDK activating kinase activity (8,10).…”

High Yield Expression of Serine/Threonine Protein Phosphatase Type 5, and a Fluorescent Assay Suitable for Use in the Detection of Catalytic Inhibitors

“…Notably, PP5 has been shown to associate with several proteins that affect signal transduction networks [1][2][3][4][5][6][14][15][16][17][18]. When PP5 is displaced from these biological partners, phosphatase activity is greatly decreased.…”

“…However, the advent of siRNA and antisense oligonucleotides capable of specifically suppressing individual phosphatases has greatly aided our understanding of the biological roles of ser/thr protein phosphatases. From such studies serine/threonine protein phosphatase type 5 (PP5) has emerged as an important regulator of hormone/stress-induced signaling networks that propagate cellular responses controlling growth and stress-induced apoptosis [1][2][3][4][5][6][7]. Nonetheless, the development of small molecule inhibitors of PP5 has been hampered because human PP5 expressed in bacteria has very low catalytic activity, and the [ 32 P]based phosphatase assays [8][9][10] commonly used to measure activity are not easily adaptable to high throughput screening assays.…”