Vikas Dhingra et al. (2001) International C. elegans Meeting "A proteomics approach to the identification of CUL-2 substrates"

Edward T Kipreos, Fernando Santiago, Vikas Dhingra, Chunling Jiang

[International C. elegans Meeting, 2001]

Cell cycle transitions are potentiated by the degradation of positive and negative cell cycle regulators through the ubiquitin-mediated proteolytic pathway. Cul-2, a component of a ubiquitin-ligase complex, regulates diverse aspects of cell cycle progression. Human Cul2 functions with the VHL tumor suppressor protein in a CUL2/VCB complex. Germline mutations in VHL cause a hereditary cancer syndrome. In vertebrates, the CUL2/VCB complex contains four protein subunits, CUL2, Rbx1, Elongin C, and VHL, and has been shown to function as a ubiquitin ligase in vitro. In C. elegans, CUL-2 negatively regulates CKI-1 and is required for the G1-to-S phase transition in germ cells. Inactivation of maternal cul-2 product results in an early embryonic arrest with only 24 cells on average. There are three major phenotypes in the cul-2 mutant embryos: a defect in chromosome condensation, leading to the formation of multiple nuclei; a delay in mitotic progression, with six fold longer mitosis than in wild type; and a defect in cytoskeletal movements. Inactivation of a ubiquitin-ligase is expected to result in the accumulation of substrates. As CUL-2 is predicted to function as a ubiquitin ligase, cul-2 mutants should have an accumulation of substrates that contribute to the observed phenotypes. We have performed a proteomic survey using two-dimensional gel electrophoresis to identify proteins whose levels change in cul-2 RNAi animals relative to wild type. In addition to the expected increase in substrate proteins, the levels of certain other proteins may also increase or decrease as a secondary consequence of an increase in the level of transcriptional regulators. These secondary changes can also be observed in a proteomic screen and can give us information about the physiological role of cul-2. We have optimized conditions for protein preparation and 2D-gel electrophoresis. We have found potential protein candidates that change in cul-2 mutants relative to wild type. These proteins will be identified by mass spectrometry, and we hope to be able to present this data at the meeting.

Vikas Dhingra et al. (2002) East Coast Worm Meeting "CUL-2 regulates the level of multiple cellular proteins"

Vikas Dhingra, Edward T Kipreos

[East Coast Worm Meeting, 2002]

Cullin/RING finger ubiquitin ligase complexes regulate a wide range of cellular processes by targeting protein degradation. In humans, the cullin CUL2 functions with the VHL tumor suppressor protein, Rbx1, and Elongin C/B in a CUL2/VCB complex. The only known substrates of the CUL2/VCB complex are the hypoxia-inducible transcription factors HIF-1 and HIF-2. In C. elegans, CUL-2 functions as a positive cell cycle regulator and is required for the G1-to-S phase cell cycle transition in germ cells by negatively regulating CKI-1 (a cyclin dependent kinase inhibitor). The three major phenotypes observed in cul-2 mutant embryos are defects in chromosome condensation, delayed mitotic progression, and defective cytoskeletal dynamics. We propose to identify CUL-2 substrates and characterize their cellular functions using a proteomics approach, thereby unraveling the molecular pathways regulating these CUL-2-dependent processes. Inactivation of a ubiquitin ligase is predicted to result in the accumulation of substrates that are no longer being degraded by the proteasome. CUL-2 is predicted to function as a ubiquitin ligase and the cul-2 mutant phenotypes may therefore arise from an accumulation of substrates. A survey of the levels of all cellular proteins in cul-2mutants compared to wild type should enable us to identify cul-2 substrates. This approach has been adopted by performing a proteomics screen using Two Dimensional Differential In Gel Electrophoresis (2D DIGE), in which mutant and wild type proteins are labeled with different Cy dyes and then run on the same gel to compare relative intensities of the dye in protein spots. Proteins whose levels change in cul-2 RNAi animals relative to wild type will be identified by mass spectrometry. When proteins are run on pH 4-7 IPG strips, we observed that five proteins have increased levels in cul-2 mutant lysates. Presently, we have identified two proteins from this screen: NADP dependent cytosolic Isocitrate Dehydrogenase and Vacuolar ATP synthase. With samples run on pH 5-6.7 IPG strips, we have observed increased levels of 11 proteins and decreased levels of 6 other proteins. We hypothesize that the decreased levels of certain proteins could be due to secondary effects resulting from altered levels of transcriptional regulators. Identification of the other CUL-2 regulated proteins is currently in progress. We are also characterizing the role of Isocitrate Dehydrogenase and Vacuolar ATP synthase in CUL-2 mediated processes.