The beta subunit of CKII negatively regulates Xenopus oocyte maturation

Abstract

CKII (formerly known as casein kinase II) is a ubiquitously expressed enzyme that plays an important role in regulating cell growth and differentiation. The beta subunit of CKII (CKIIbeta) is not catalytic but forms heterotetramers with the catalytic subunit alpha to generate an alpha2beta2 holoenzyme. In Xenopus oocytes, CKIIbeta also associates with another serine/threonine kinase, Mos. As a key regulator of meiosis, Mos is necessary and sufficient to initiate oocyte maturation. We have previously shown that the binding of CKIIbeta to Mos represses Mos-mediated mitogen-activated protein kinase (MAPK) activation and that the ectopic expression of CKIIbeta inhibits progesterone-induced Xenopus oocyte maturation. We have now used an antisense oligonucleotide technique to reduce the endogenous CKIIbeta protein level in Xenopus oocytes, and we find that oocytes with a reduced content of CKIIbeta are more sensitive to low doses of progesterone and show accelerated MAPK activation and germinal vesicle breakdown. Furthermore, ectopic expression of a Mos-binding fragment of CKIIbeta suppressed the effect of antisense oligonucleotide. These results suggest that the endogenous CKIIbeta normally sets a threshold level for Mos protein, which must be exceeded for Mos to activate the MAPK signaling pathway and induce oocyte maturation.

Figure 1

The effect of antisense and sense oligonucleotides on CKIIβ protein level. Xenopus CKIIβ antisense and sense oligonucleotides (60–80 ng) were injected into stage VI Xenopus oocytes. O, uninjected oocyte; E, uninjected egg; S, sense oligonucleotide; AS, antisense oligonucleotide. (A) The injected oocytes were treated with 500 ng/ml progesterone for 8 h and analyzed by anti-CKIIβ or anti-MAPK immunoblotting. CKII prepared from recombinant baculovirus was used as a positive control. (B) Oocytes were coinjected with Myc-CKIIβ mRNA (1 mg/ml) mixed with H₂O, antisense, or sense oligonucleotides. After incubation at room temperature for 4 h, 50 ng/ml progesterone was added and the incubation was continued for 12 h. Myc-CKIIβ protein was detected by immunoblotting duplicate samples. —, control oocytes lacking Myc-CKIIβ.

Figure 2

CKIIβ antisense oligonucleotide promoting oocyte maturation. The antisense and sense oligonucleotides were injected into oocytes followed by progesterone treatment at various concentration for 8 h. (A) The percentage of GVBD was scored. (B) Oocyte extracts were prepared from A and analyzed by anti-MAPK immunoblotting, myelin basic protein (MBP) kinase assay, histone H1 kinase assay, as well as anti-Mos immunoblotting.

Figure 3

CKIIβ antisense oligonucleotide accelerating MAPK phosphorylation and GVBD. A batch of oocytes was injected with oligonucleotides and treated with progesterone for various times. (A) Five oocytes were taken and frozen in liquid N₂ for further analysis by anti-MAPK or anti-phospho-MAPK immunoblotting. (B) Progesterone (50 ng/ml) was added to the injected oocytes, and GVBD was scored at the indicated times.

Figure 4

The rescue of CKIIβ antisense oligonucleotide effect by GST-CKIIβ_141–215. Twelve hours after the oligonucleotide injection, oocytes were reinjected with GST or GST-CKIIβ_141–215 protein followed by progesterone treatment. (A) The percentage of GVBD was scored 8 h later. (B) Oocyte extracts were prepared from A and analyzed by anti-MAPK immunoblotting. G, GST; β, GST-CKIIβ_141–215.

Figure 5

A proposed model for the CKIIβ function during Xenopus oocyte maturation. The initiation of oocyte maturation can be divided into three steps. Progesterone stimulates Mos protein synthesis. The newly synthesized Mos protein (represented as a box) binds to CKIIβ (represented as a circle) and is inactive (step 1). As meiosis progresses, the amount of Mos protein reaches and exceeds that of CKIIβ (step 2). Free Mos molecules are active and can phosphorylate MKK. The activated MKK goes on to activate its substrate, MAPK. Activated MAPK is important for MPF activation and for further stimulating Mos protein synthesis. The activation of MPF leads to GVBD (step 3).