Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2

Abstract

We used the interaction trap, a yeast genetic selection for interacting proteins, to isolate human cyclin-dependent kinase interactor 1 (Cdi1). In yeast, Cdi1 interacts with cyclin-dependent kinases, including human Cdc2, Cdk2, and Cdk3, but not with Ckd4. In HeLa cells, Cdi1 is expressed at the G1 to S transition, and the protein forms stable complexes with Cdk2. Cdi1 bears weak sequence similarity to known tyrosine and dual specificity phosphatases. In vitro, Cdi1 removes phosphate from tyrosine residues in model substrates, but a mutant protein that bears a lesion in the putative active site cysteine does not. Overexpression of wild-type Cdi1 delays progression through the cell cycle in yeast and HeLa cells; delay is dependent on Cdi1 phosphatase activity. These experiments identify Cdi1 as a novel type of protein phosphatase that forms complexes with cyclin-dependent kinases.

Bcl-2 family members inhibit oxidative stress-induced programmed cell death in Saccharomyces cerevisiae

“…The cDNAs encoding human bcl-2 (obtained from S. Korsmeyer, Dana-Farber Cancer Institute, Boston [29]), chicken bcl-xl (obtained from C. Thompson, University of Chicago [30]) and C. elegans ced-9 (obtained from R. Horvitz, Massachusetts Institute of Technology, Cambridge [31]) were initially cloned between the Eco RI and Xho I restriction endonuclease cut sites in the yeast expression plasmid, pEG202 [32], which carries the S. cerevisiae HIS3 gene as a selectable marker. The following primers were used for Bcl-2: 5'-GGTGAATTCATGGCGCACGCTGGGAGA-ACA-3' (forward) and 5'-CCTCTCGAGTCACTT-GTGGCCCAGATAGGC-3' (reverse); for Bcl-xL: 5'-GGTGAATTCATGTCTCAGAGCAACCGGGAG-3' (forward) and 5'-CCTCTCGAGTCATTTCCGACT-GAAGAGTGA-3' (reverse); for CED-9:…”

p53 is involved in the p120E4F-mediated growth arrest

“…The yeast two-hybrid screen (Gyuris et al, 1993) was employed to identify proteins able to associate with the p53H175 hot spot mutant. Approximately 3.5 million independent transformants were screened and 27 single clones were isolated and analysed for their ability to bind also wt p53 (LexAwtp53, aa 74-393).…”

“…To generate the LexA-fusion constructs, human p53H175 and wt p53 cDNAs were PCR-ampli®ed between aa 74 and aa 393 and cloned in-frame into pLexA202 (Gyuris et al, 1993). To construct pcDNA3HAE4FD350, the E4F cDNA (from aa 350 to aa 783), isolated from the two-hybrid screening was inserted into pcDNA3 (Invitrogen) downstream to a START codon and a contiguous HA-epitope.…”

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

“…The budding yeast strains used for the two-hybrid screens were EGY48: a, URA3, 6LexAop LEU2, his3-, trp1-, URA3::pSH1834 [30], and Mav203: a, ade2-101, ura3D, leu2-3, lys2-801, trp1-D63, Dgal4 Dgal80, SPAL::URA3, GAL1-LacZ, GAL1-HIS3 [31]. The Mcs2 yeast two-hybrid screen was performed essentially as described [32] using Mcs2-pEG202 as bait against a fission yeast cDNA library (a kind gift of V. Damagnez) in pJG4-5. The Skp1 two-hybrid was performed according to manufacturerÕs protocol (Matchmaker, Clontech) using Skp1-pPC87 against a fission yeast cDNA library (a kind gift of S. Elledge) in pACT2.…”