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Sci. Signal., 3 November 2009
Vol. 2, Issue 95, p. ec353
[DOI: 10.1126/scisignal.295ec353]


Biochemistry Self-Activating, but Still Regulated

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

The transition from egg to embryo is complex and involves two stages. In the nematode Caenorhabditis elegans, an unusual dual specificity kinase MBK-2, a member of the DYRK family, plays a crucial role in the egg-to-embryo transition by regulating the activity of proteins involved in meiosis. DYRK are self-activating kinases that become autophosphorylated on a tyrosine residue during translation and then serve as serine/threonine kinases. MBK-2 is sequestered in the cytosol before meiotic division by the interaction of the pseudophosphatase EGG-3 with a region of MBK-2 outside the catalytic domain, and release of MBK-2 requires the activity of cyclin-dependent kinase 1 (CDK-1) and the ubiquitin ligase APC/C. Cheng et al. now show that the activity of MBK-2 during meiosis is stimulated by phosphorylation by CDK-1 and is inhibited by two closely related pseudophosphatases, EGG-4 and EGG-5 (EGG-4/5), which act as substrate-trapping molecules, binding, but not dephosphorylating, MBK-2 at the autophosphorylated tyrosine residue and preventing MBK-2 from phosphorylating its substrates. In vitro kinase assays showed that MBK-2 (a catalytically inactive mutant) was phosphorylated by CDK-1 on Ser68. In adult worms, phosphorylated MBK-2 was detected only in mature hermaphrodites, which have both oocytes and embryos, but not in young adults, which contain only oocytes but no embryos. Furthermore, a S68E phosphorylation mimicking mutant MBK-2, but not a S68A mutant, rescued mbk-2 mutants. When CDK-1 was knocked down, phosphorylation of an MBK-2 substrate MEI-1 was reduced in worms expressing wild-type MBK-2 but not in worms expressing the S68E mutant. Unexpectedly, phosphorylation by CDK-1 did not appear to directly stimulate the kinase activity of MBK-2, because when expressed and immunoprecipitated from mammalian cells and then exposed to hCDK-1, MBK-2 and the S68A mutant exhibited similar activities toward recombinant MEI-1, and when produced as recombinant proteins in bacteria, wild-type MBK-2, S68A, and S68E all showed similar catalytic activity. Thus, CDK-1 phosphorylation likely promotes the interaction of MBK-2 with other regulatory molecules to stimulate MBK-2 activity during meiosis. The authors investigated MBK-2 activity in oocytes of worms deficient for EGG-4/5 and found that phosphorylated MEI-1 was present in the most mature oocytes; when EGG-4/5 were knocked down in the presence of S68E MBK-2, however, phosphorylated MEI-1 was also present in less mature oocytes. Because EGG-4/5, like MBK-2, are released into the cytosol in worms deficient for EGG-3, the author postulated that EGG-4/5 did not inhibit MBK-2 through a sequestration mechanism. The ability of EGG-3 and EGG-4/5 required their pseudophosphatase active sites, but EGG-3 bound to MBK-2 at the N terminus and did not require the ATP-binding site of MBK-2. In contrast, EGG-4/5 bound MBK-2 at the active site and required at least one of the tyrosines at the MBK-2 active site and the MBK-2 ATP-binding site. These interactions were confirmed in worms by colocalization and coimmunoprecipitation analysis. EGG-4 inhibited MBK-2 activity in an in vitro kinase assay without altering the tyrosine phosphorylation state of MBK-2. Kinetic analysis suggested that EGG-4 altered the Km and the Vmax of the activity of MBK-2 for MEI-1. Thus, the self-activating kinase MBK-2 uses CDK-1 phosphorylation to stimulate activity in specific contexts and pseudophosphatases to inhibit activity through two mechanisms, sequestration and direct inhibition of the kinase (see commentary by Tonks).

N. K. Tonks, Pseudophosphatases: Grab and hold on. Cell 139, 464–465 (2009). [Online Journal]

K. C.-C. Cheng, R. Klancer, A. Singson, G. Seydoux, Regulation of MBK-2/DYRK by CDK-1 and the pseudophosphatases EGG-4 and EGG-5 during the oocyte-to-embryo transition. Cell 139, 560–572 (2009). [Online Journal]

Citation: N. R. Gough, Self-Activating, but Still Regulated. Sci. Signal. 2, ec353 (2009).

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