Self-Activating, but Still Regulated

Science Signaling  03 Nov 2009:
Vol. 2, Issue 95, pp. ec353
DOI: 10.1126/scisignal.295ec353

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]