Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation

Lethality of mice bearing a knockout of the <i>Ngly1</i>-gene is partially rescued by the additional deletion of the <i>Engase</i> gene

<div><p>The cytoplasmic peptide:<i>N</i>-glycanase (Ngly1 in mammals) is a de-<i>N</i>-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the <i>NGLY1</i> gene exhibited severe systemic symptoms (<i>NGLY1</i>-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed <i>Ngly1</i>-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-<i>N</i>-acetylglucosaminidase (<i>Engase</i>), which is another de-<i>N</i>-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the <i>Ngly1</i>-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of <i>Ngly1</i>-deficient mice. Viable <i>Ngly1</i>-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of <i>NGLY1</i>-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of <i>Engase</i> in the C57BL/6 and ICR mixed background. The defects observed in <i>Ngly1/Engase</i>-deficient mice (C57BL/6 background) and <i>Ngly1</i>-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an <i>NGLY1</i>-deficiency. These observations strongly suggest that the <i>Ngly1</i>- or <i>Ngly1/Engase</i>-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the <i>NGLY1-</i>deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.</p></div