A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.

B G Gabrielli,L M Roy,J Gautier,M Philippe,J L Maller

doi:10.1016/s0021-9258(18)46041-3

B G Gabrielli, L M Roy + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)46041-3

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Abstract

The Eg1 gene in Xenopus laevis is related in sequence to the cdc2+ gene. We show here that the Eg1 gene product (cdk2) possesses histone H1 protein kinase activity and binds to PSTAIR antibodies as well as to Sepharose beads linked to the 13-kDa product of the suc 1 gene (p13suc1). Eg1 protein kinase is active only in an Mr approximately 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro. The protein kinase activity of Eg1 oscillates in the mitotic cell cycle, being high in M-phase and low in interphase. Hyperactivation of cdc2 kinase by the addition of cyclin A has no effect on the activity or oscillatory behavior of Eg1. Inhibition of cdc2 kinase activation by emetine or RNase treatment of oscillating extracts does not inhibit the activation of Eg1 but does block deactivation normally seen during exit from mitosis. These results indicate that Eg1 is regulated by a cell cycle clock independently of cyclin and cdc2 kinase.

Highlights

The Egl gene inXenopus laevis is related insequence In activated Xenopus egg extracts which oscillate between M
We show here that the E g l gene and S-phase, the destruction of cyclin is required for exit from product possesses histone H1 protein kinase mitosis and leads to inactivation of cdc2 kinase [9]
Egplrotein kinase is active only in an M,= 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro

Summary

MATERIALS AND METHODS

Control SepharoseCL-4B, protein A-Sepharose, The first oligonucleotide was homologous to the firstATG region of and pl3-Sepharose were incubated with 10 mg/ml BSA prior to use, the gene containing an NcoI site, and thesecond oligonucleotide was t o decrease nonspecific binding to the Sepharosebeads. Preparation ojpl3-Sepharose-pl3””’ was purified from a n over- contained an XbaI site 3’ of the homologous sequence. This fragment producing strain of Escherichia coli by gel filtration on Sepharose was subcloned into the pEPEX vector’ between the NcoI and CL-GB (Pharmacia) as described in Brizuela et al [17]. For immunoblotting experiments,a 0-34% am- uitro translation of the capped mRNAwas done in reticulocyte lysates monium sulfate fraction of a Xenopus egg extract was diluted with (Promega) according to themanufacturer’s instructions. E B but notprecleared prior to precipitation with pl3-Sepharose

RESULTS

Findings

DISCUSSION