Maternal Xenopus Cdk2-Cyclin E Complexes Function during Meiotic and Early Embryonic Cell Cycles That Lack a G1 Phase

Rachel E Rempel,Susan B Sleight,James L Maller

doi:10.1074/jbc.270.12.6843

Rachel E Rempel, Susan B Sleight + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.12.6843

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Abstract

Earlier work demonstrated that cyclins A1, B1, and B2 are not associated with Cdk2 from unfertilized Xenopus eggs. As a potential Cdk2 partner during meiosis, a cyclin E homolog was cloned from a Xenopus oocyte cDNA library and found to be 60% identical at the amino acid level to human cyclin E. Cyclin E1 protein was detected in resting oocytes, and the level increased severalfold in meiosis II, concomitant with the appearance of forms with decreased electrophoretic mobility. During oocyte maturation, the patterns of cyclin E1-associated kinase activity and Cdk2 activity were identical, with activity low until after germinal vesicle breakdown, peaking during meiosis II. Cyclin E1 complexes immunoprecipitated from unfertilized Xenopus eggs contained Cdk2 but not Cdc2. In cycling egg extracts Cdk2-cyclin E1-associated kinase activity oscillated, but the level of cyclin E1 protein and its association with Cdk2 did not vary appreciably; complex activity appeared to be regulated neither by the synthesis and destruction of the cyclin subunit nor by association/disassociation of the two subunits. During the early cleavage divisions in embryos, cyclin E1 and Cdk2 remained associated. The data indicate that the Cdk2-cyclin E complex functions during meiotic and embryonic cell cycles in addition to performing its established role during G1 in somatic cells.

Highlights

From the Howard Hughes Medical Institute and Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262
Earlier work demonstrated that cyclins AI, BI, and B2 are not associated with Cdk2 from unfertilized Xenopus eggs
Three cell cycle regulators, maturation-promoting factor (MPF),1 cytostatic factor (C8F), and Cdk2 were discovered in Xenopus oocytes and eggs [10, 11]

Summary

EXPERIMENTAL PROCEDURES

Preparation of Oocytes, Egg Extracts, and Embryos-Stage VI 00cytes were manually dissected from their follicular envelopes and induced to mature by addition of progesterone to 10 IJoM. Phosphatase Treatment of Cyclin El Immunoprecipitates-Endogenous cyelin E1 was immunoprecipitated from a CSF extract arrested in metaphase of meiosis II using rabbit immune serum These immunoprecipitates were washed as for kinase assays using buffers supplemented with 50 mM NaF, 30 mMp-nitrophenyl phosphate, and 0.5 mM phenylmethylsulfonyl fluoride. Radiolabeled cyelin E1 was recovered using affinity-purified goat immune serum and protein G-Sepharose; some sample was treated with antibody blocked by prior incubation with recombinant cyclin EI. Prior to treatment with phosphatase, both sets of immunoprecipitates were washed twice in a phosphatase buffer (50 mM Tris-HCl, pH 7.4,30 mMNaCl, 1 mMEDTA, 2 mMDTT, 0.5 IDM phenylmethylsulfonyl fluoride, or 200 J.LM Pefabloc, and 10 J.Lg/ml each aprotinin, leupeptin, chymostatin, and pepstatin), Endogenous cyclin E1 immunoprecipitates were incubated for 30 min at 23°C with either buffer, recombinant X. laevis PP1-y1, purified PP2A Endogenous eyclin E1 was visualized by irnmunoblotting with blot-purified goat antibody to cyelin E1, whereas radiolabeled cyelin E1 was visualized by autoradiography

RESULTS

F MTVGII

Autoradioqraph

IP-Western