Biochem. J. (2003) 373, 583–591 (Printed in Great Britain) 583
Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1 Sopheap PHIN1,Deborah KUPFERWASSER1, Joseph LAM and Kay K. LEE-FRUMAN2 Department of Biological Sciences, California State University at Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840, U.S.A.
Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). Thr-388. MEK-dependent phosphorylation of the autoinhibitory S6K1 and S6K2 show different cellular localization as well serines in S6K2 occurs prior to Thr-388 activation. Combining as divergent amino acid sequences in non-catalytic domains, T388E and T228A mutations inhibited S6K2 activation, and suggesting that their cellular functions and/or regulation may not akinase-inactive phosphoinositide-dependent protein kinase be identical. Many of the serine/threonine residues that become (PDK1) diminished T388E activity, suggesting that the role phosphorylated and contribute to S6K1 activation are conserved of Thr-388 is to allow further phosphorylation of Thr-228 by in S6K2. In this study we carry out mutational analyses of PDK1. Thr-388 fails to become phosphorylated in Ser-370 these serine/threonine residues on S6K2 in order to elucidate the mutants, suggesting that the role of Ser-370 phosphorylation mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 may be to allow Thr-388 phosphorylation. Finally, using the are crucial for S6K2 activity, and the three proline-directed serines rapamycin-resistant T388E mutant, we provide evidence that in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play S6K2 can phosphorylate S6 in vivo. aroleinS6K2activityregulation in a mitogen-activated pro- tein kinase/extracellular-signal-regulated kinase kinase (MEK)- dependent manner. However, unlike S6K1, changing Thr-388 Key words: mammalian target of rapamycin (mTOR), p70 to glutamic acid in S6K2 renders the kinase fully active. This ribosomal S6 kinase 1 (S6K1), phosphoinositide 3-kinase (PI3K), activity was resistant to the effects of rapamycin or wortmannin, phosphoinositide-dependent protein kinase (PDK1), ribosomal S6 indicating that mammalian target of rapamycin (mTOR) and kinase 2 (S6K2).
INTRODUCTION S6K2 was cloned based on its nucleotide sequence homology to S6K1, and its cellular function and in vivo substrate(s) are Phosphorylation of the 40 S ribosomal protein S6 is one of the not yet established. Initial studies on S6K2 showed that it can events that occur during cell proliferation in many different cell phosphorylate S6 in vitro,andthatupstream kinases that have types under various mitogenic conditions. S6 phosphorylation been shown to regulate the catalytic activity of S6K1, such as results in up-regulation of translation of mRNA species that phosphoinositide 3-kinase (PI3K), phosphoinositide-dependent contain 5 -terminal oligopyrimidine tract, many of which encode protein kinase (PDK1), mTOR, Cdc42, Rac and protein kinase proteins that are themselves involved in translation [1]. This in turn C ζ ,may also play a role in S6K2 regulation [11–16]. The increases the cell’s protein synthesis capability in preparation for current hypothesis is that S6K1 and S6K2 may have common cell proliferation. p70 Ribosomal S6 kinase 1 (S6K1), previously as well as distinct functions in cells. This is based on several known as p70 S6 kinase, is a serine/threonine kinase responsible lines of evidence. Although mouse embryonic fibroblasts from for S6 phosphorylation. The importance of S6K1 in cellular S6K1-knockout mice show normal proliferation and normal S6 proliferation was underscored when it was discovered that a phosphorylation in vivo,the mice had a small-body phenotype due potent mitogenic inhibitor and immunosuppressant rapamycin to defects in cell size and growth regulation [11]. This suggests might exert its function by inhibiting the activity of S6K1 that S6K2 may be able to carry out some but not all of the [2–6]. However, rapamycin does not inhibit S6K1 directly; functions of S6K1 in the absence of S6K1. Amino acid sequence rather, rapamycin binds FK506-binding protein 12 (FKBP12), comparison between the two kinases shows diverging sequences and this complex in turn binds and inhibits mammalian target in non-catalytic regions, hinting at differential function and/or of rapamycin (mTOR) [7]. mTOR plays a role as a nutrient regulation. S6K1 has two isoforms that arise from alternative sensor in cellular proliferation, and it controls not only S6K1 translational start sites that differ by 23 amino acids in the N- butalso other proteins, including initiation factor 4E-binding terminus (αIandαII) [17,18]. The nuclear localization signal protein [8–10]. Interestingly, cells derived from mice lacking for S6K1 resides in this 23 amino acid N-terminal portion, and S6K1 showed no defect in cellular proliferation and no defect therefore the αII isoform is found mainly in the cytoplasm whereas in S6 phosphorylation in vivo [11]. These data suggest that there the longer αIisoform resides in the nucleus [19,20]. S6K2 also may exist additional kinase(s) that can substitute for S6K1. This has two isoforms with alternative translational start sites (βI has led to identification and cloning of ribosomal S6 kinase 2 and βII), but the nuclear localization signal for S6K2 is in the (S6K2) [11–15]. C-terminal portion of the protein and therefore both isoforms
Abbreviations used: S6K2, ribosomal S6 kinase 2; S6K1, p70 ribosomal S6 kinase 1; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; PDK1, phosphoinositide-dependent protein kinase; MEK, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase; EGF, epidermal growth factor; HA, haemagglutinin; DTT, dithiothreitol; Ab, antibody. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (e-mail [email protected]).