Abstract
During mitosis, the cyclin-dependent kinase, Cdc2, signals the inactivation of major anabolic processes such as transcription, mRNA processing, translation, and ribosome biogenesis, thereby providing energy needed for the radical and energetically costly structural reorganization of the cell. This is accomplished by phosphorylation and inactivation of several key anabolic elements, including TFIIIB, TFIID, RNA polymerase II, poly(A) polymerase, and translation elongation factor 1gamma. We report here that ribosomal S6 kinase 1 (S6K1), a protein kinase linked to the translation of ribosomal protein mRNAs, is also subject to regulation by Cdc2 in mitosis. In mitotic HeLa cells, when the activity of Cdc2 is high, S6K1 is phosphorylated at multiple Ser/Thr, Pro (S/TP) sites, including Ser(371), Ser(411), Thr(421), and Ser(424). Concomitant with this, the phosphorylation of the hydrophobic motif site, Thr(389), is reduced resulting in a decrease in the specific activity of S6K1. The mitotic S/TP phosphorylation sites are readily phosphorylated by Cdc2.cyclin B in vitro. These proline-directed phosphorylations are sensitive to chemical inhibitors of Cdc2 but not to inhibitors of mammalian target of rapamycin, phosphatidylinositol 3-kinase, MEK1/2, or p38. In murine FT210 cells arrested in mitosis, conditional inactivation of Cdc2 reduces phosphorylation of S6K1 at S/TP sites while simultaneously increasing phosphorylation of Thr(389) and of the S6K1 substrate, RPS6. A physical interaction exists between Cdc2 and S6K1, and this interaction is enhanced in mitotic cells. These results suggest that Cdc2 provides a signal that triggers inactivation of S6K1 in mitosis, presumably serving to spare energy for costly mitotic processes at the expense of ribosomal protein synthesis.
Highlights
During the mitotic phase of the cell cycle, the substructure of mammalian cells is reorganized through a tightly regulated series of events, which ensures that each of the two resulting daughter cells receives the appropriate complement of genetic material
We have demonstrated that S6 kinase 1 (S6K1) is phosphorylated on both consensus and non-consensus S/TP residues in vitro by Cdc21⁄7cyclin B and in vivo in a mitosisspecific fashion
Mitotic phosphorylation of S6K1 is associated with its inactivation as evidenced by decreased specific kinase activity and dephosphorylation of its substrate, RPS6
Summary
S6K1, ribosomal S6 kinase 1; PDK1, phosphoinositide-dependent kinase 1; S/TP, Ser/Thr, Pro sites; mTOR, mammalian target of rapamycin; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ ERK kinase; JNK, c-Jun N-terminal kinase; FACS, fluorescence-activated cell sorting. A requirement for phosphorylation of Ser371, an additional S/TP site removed from the S/TP cluster within the autoinhibitory domain, for kinase activity has been demonstrated [18], its order in the hierarchy of serum-stimulated phosphorylations is unclear. Ser411 lies within a strong consensus for Cdc phosphorylation (K/RpSPR/PR/K/H [23]) and is phosphorylated during mitosis when the activity of Cdc is augmented [9]. Many Cdc substrates possess multiple S/TP sites, not all of which conform to the consensus sequence of phosphorylation, e.g. lamins [31], Src [32], eIF4E binding protein 1 [33], and poly(A) polymerase [5]. We demonstrate that Cdc is required for mitotic inactivation S6K1 in FT210 cells
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