Dataset Information

Human CDK Family - Proteomic Analysis of the Human CDK Family Reveals a Novel CDK5 Complex Involved in Cell Growth and Migration

ABSTRACT: Cyclin-dependent kinases (Cdks) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in control of cell-cycle progression, transcription and neuronal functions. To systematically investigate the regulations and functions of CDKs, we conducted a proteomic analysis of human CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. We discovered 753 HCIPs (high-confidence candidate interaction proteins) in HEK293T cells and 352 HCIPs in MCF10A cells.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Mcf-10a Cell, Permanent Cell Line Cell, Hek-293t Cell

SUBMITTER: Xu Li

LAB HEAD: Junjie Chen

PROVIDER: PXD000593 | Pride | 2014-08-08

REPOSITORIES: Pride

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Publications

Proteomic analysis of the human cyclin-dependent kinase family reveals a novel CDK5 complex involved in cell growth and migration.

Xu Shuangbing S Li Xu X Gong Zihua Z Wang Wenqi W Li Yujing Y Nair Binoj Chandrasekharan BC Piao Hailong H Yang Kunyu K Wu Gang G Chen Junjie J

Molecular & cellular proteomics : MCP 20140805 11

Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines ...[more]

PMID: 25096995

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Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

Dataset Information

Human CDK Family - Proteomic Analysis of the Human CDK Family Reveals a Novel CDK5 Complex Involved in Cell Growth and Migration

Publications

Proteomic analysis of the human cyclin-dependent kinase family reveals a novel CDK5 complex involved in cell growth and migration.

Similar Datasets

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