Measuring APC/C-Dependent Ubiquitylation In Vitro.

The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

Phosphorylation of mixed lineage leukemia 5 by CDC2 affects its cellular distribution and is required for mitotic entry.

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G(2)/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G(2) phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.

cdc2-cyclin B regulates eEF2 kinase activity in a cell cycle- and amino acid-dependent manner.

The calcium/calmodulin-dependent kinase that phosphorylates and inactivates eukaryotic elongation factor 2 (eEF2 kinase; eEF2K) is subject to multisite phosphorylation, which regulates its activity. Phosphorylation at Ser359 inhibits eEF2K activity even at high calcium concentrations. To identify the kinase that phosphorylates Ser359 in eEF2K, we developed an extensive purification protocol. Tryptic mass fingerprint analysis identified it as cdc2 (cyclin-dependent kinase 1). cdc2 co-purifies with Ser359 kinase activity and cdc2-cyclin B complexes phosphorylate eEF2K at Ser359. We demonstrate that cdc2 contributes to controlling eEF2 phosphorylation in cells. cdc2 is activated early in mitosis. Kinase activity against Ser359 in eEF2K also peaks at this stage of the cell cycle and eEF2 phosphorylation is low in mitotic cells. Inactivation of eEF2K by cdc2 may serve to keep eEF2 active during mitosis (where calcium levels rise) and thereby permit protein synthesis to proceed in mitotic cells. Amino-acid starvation decreases cdc2's activity against eEF2K, whereas loss of TSC2 (a negative regulator of mammalian target of rapamycin complex 1(mTORC1)) increases it. These data closely match the control of Ser359 phosphorylation and indicate that cdc2 may be regulated by mTORC1.

Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle.

ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis in budding yeast. Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth. Second, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; because of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize the actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. Previous results indicate a connection between Rot1 and the cyclin Clb2. In fact, overexpression of CLB2 is toxic when ROT1 is partially inactivated, and reciprocally, deletion of CLB2 suppresses the lethality of the rot1 mutant, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and we show that the Clb2 cyclin is not properly degraded in rot1 cells.

The crystal structure of human cyclin B.

Cyclin B is the key regulatory protein controlling mitosis in all eukaryotes, where it binds cyclin-dependent kinase, cdk1, forming a complex which initiates the mitotic program through phosphorylation of select proteins. Cyclin B regulates the activation, subcellular localization, and substrate specificity of cdk1, and destruction of cyclin B is necessary for mitotic exit. Overexpression of human cyclin B1 has been found in numerous cancers and has been associated with tumor aggressiveness. Here we report the crystal structure of human cyclin B1 to 2.9 A. Comparison of the structure with cyclin A and cyclin E reveals remarkably similar N-terminal cyclin box motifs but significant differences among the C-terminal cyclin box lobes. Divergence in sequence gives rise to unique interaction surfaces at the proposed cyclin B/cdk1 interface as well as the 'RxL' motif substrate binding site on cyclin B. Examination of the structure provides insight into the molecular basis for differential affinities of protein based cyclin/cdk inhibitors such as p27, substrate recognition, and cdk interaction.

Enzymology of the anaphase-promoting complex.

The anaphase-promoting complex (APC) is an ubiquitin-protein ligase that promotes mitotic progression by catalyzing the ubiquitination of numerous proteins, including securin and cyclin. Its complex subunit composition and extensive regulation make the APC an active subject of investigation for both cell biologists and enzymologists. This chapter describes a system for the reconstitution and quantitative analysis of APC activity from budding yeast in vitro. We focus in particular on the measurement of processive ubiquitination, which complements traditional analysis of the reaction rate as a means to elucidate the molecular details of substrate recognition and ubiquitination by the APC.

Measurement of Wee kinase activity.

The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activity of the Wee kinases are regulated during the cell cycle and development. In this chapter, we describe several procedures to measure the activity of the Wee kinases found either in crude extracts or in purified preparations. Specific protocols include the production and purification of recombinant Cdc2/Cyclin B substrate, the production of crude subcellular extract fractions, the purification of endogenous or recombinant Wee kinases, Wee kinase assays, and the Histone H1 kinase assay to measure Cdc2 activity. In addition, support protocols are provided that describe the use and production of Ni-IDA beads for the purification of Histidine-tagged proteins, and the use of the baculovirus expression system to produce recombinant proteins.

A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Cleavage of cyclin A at R70/R71 by the bacterial protease OmpT.

Previous work has shown that cyclin A can be cleaved at Arg-70/Arg-71 by a proteolytic activity present in an in vitro-coupled transcription/translation system by using rabbit reticulocyte lysate programmed by plasmid DNA encoding p27(KIP1), a cyclin-dependent kinase inhibitor, but not by plasmid DNAs encoding other cyclin-dependent kinases inhibitors. Here we report that cyclin A is also cleaved by translation product programmed by plasmid DNA encoding cyclin B. Several findings indicate that the cleavage activity in this assay is provided by the bacterial protease OmpT, which cofractionates with cyclin B and p27(KIP1) plasmid DNAs and is thus carried over into the coupled in vitro transcription/translation reactions. (i) Cleavage activity appeared even when transcription or translation of the cyclin B or p27(KIP1) was blocked. (ii) Activity resembling OmpT, a serine protease that cleaves between dibasic residues, routinely copurifies with p27(KIP1) and cyclin B plasmid DNAs. (iii) Both cyclin A cleavage activity and OmpT activity are heat stable, resistant to denaturation, and inhibited by Zn(2+), Cu(2+), or benzamidine. (iv) Cyclin A cleavage activity is detected when using lysates or DNAs prepared from Escherichia coli strains that contained OmpT but not with strains lacking OmpT. (v) Purified OmpT enzyme itself cleaves cyclin A at R70/R71. These data indicate that OmpT can be present in certain DNA preparations obtained by using standard plasmid purification protocols, and its presence can potentially affect the outcome and interpretation of studies carried out using in vitro-translated proteins.

Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes.

M-Phase promoting factor (MPF) is a complex of p34(cdc2) and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34(cdc2), rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34(cdc2) is in excess to that of cyclin B, i.e., the accumulation of p34(cdc2) is not a limiting factor. We report measurements of the absolute mass of p34(cdc2) and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 10(6), 11.3 x 10(6), 24.6 x 10(6), 15. 6 x 10(6), respectively, and in meiotically competent oocytes the numbers are 14.3 x 10(6), 95.5 x 10(6), 80.0 x 10(6), 40.1 x 10(6), respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34(cdc2), and this is consistent with the hypothesis that the accumulation of p34(cdc2) plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34(cdc2) in both meiotically incompetent and competent oocytes.

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis.

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.