Inhibition of G1/S transition in HEK293 cells by Speedy/Ringo C Free

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In addition to cyclins, cyclin-dependent kinases (CDKs) can be activated by novel cell cycle regulators termed Speedy/Ringo (S/R) proteins, which show no apparent similarity to cyclins [1-2]. In mammals, there are at least four different Speedy/Ringo proteins [4-5]. S/R proteins share a highly conserved ~140-aa region that is essential for CDK binding. Speedy/Ringo A and C bind to Cdc2 and Cdk2, whereas S/R B binds preferentially to Cdc2. Despite their distinct CDK-binding preferences, both S/R A and B can promote the maturation of Xenopus oocytes and all three S/R proteins can bind to and activate CDKs in vivo. The existence of this growing family of CDK activators suggests that S/R proteins may play as complex a role in cell cycle control as the diverse family of cyclins.

Speedy/Ringo proteins are essential for the G2/M transition during Xenopus oocyte maturation [1-2]. A human homolog (Spy1, here called S/R A1) promoted S-phase entry and cell survival after DNA damage in cultured somatic cells [3]. In this study, we investigate the biological function of Speedy/Ringo C (S/R-C). S/R-C mRNA is detected in diverse tissues, particularly those that undergo polyploidization including bone marrow, liver, kidney, and placenta. S/R-C is endogenously expressed in cell lines derived from these tissues (i.e. HEK293 and K562). Knock-down of S/R-C in HEK293 cells by RNAi led to a reduction of G1 phase cells and an increase of cells in S and G2/M. This change of cell cycle distribution can be rescued by introduction of RNAi-insensitive S/R-C into cells. Overexpression of S/R-CWT results in a dose-dependent cell growth inhibition, cell death, and G1 arrest. When they were released from double thymidine block, stable cell lines expressing S/R-CWT showed a delay of S-phase entry. The inhibitory effects of S/R-C on cell proliferation are dependent on CDK-binding because cells grow normally when S/R-CY95A, a CDK-binding defective mutant, is expressed. Thus, Speedy/Ringo C is an inhibitor of the G1/S transition acting through CDKs. How Speedy/Ringo C controls the G1/S transition is currently under investigation.

References

1.Lenormand JL, Dellinger RW, Knudsen KE, Subramani S, Donoghue DJ. EMBO J 1999; 18:1869-77.

2.Ferby I, Blazquez M, Palmer A, Eritja R, Nebreda AR. Genes Dev 1999; 13:2177-89.

3.Porter LA, Dellinger RW, Tynan JA, Barnes EA, Kong M, Lenormand JL, Donoghue DJ. J Cell Biol 2002; 157:357-66.

4.Dinarina A, Perez LH, Davila A, Schwab M, Hunt T, Nebreda AR. Biochem J. 2005; 386(Pt 2):349-55

5.Cheng A, Xiong W, Ferrell JE Jr, Solomon MJ. Cell Cycle. 2005; 4(1):155-65

6.Cheng A, Gerry S, Kaldis P, Solomon MJ. BMC Biochem. 2005; 6:19.