Inactivation of oncoprotein binding by a single Cys<sup>706</sup>‐to‐Tyr substitution in the retinoblastoma protein

Saijo, Masafumi; Kato, Mitsuo; Sasaki, Masao S.; Ishizaki, Kanji

doi:10.1016/0014-5793(94)80133-9

Cited by 4 publications

(2 citation statements)

References 29 publications

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“…Leu was substituted for Ser780 in wild-type GST-RB (pGEXRB;Saijo et al, 1994) using site-directed mutagenesis kit (Promega). The wild-type GST-RB and GST-RB-S780L proteins were produced in E.coli treated with 1 mM IPTG, and purified using glutathione-Sepharose CL-4B (Pharmacia) as described by Saijo et al (1994).…”

Section: Methodsmentioning

confidence: 99%

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

et al. 1996

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Cyclin D‐Cdk4/6 and cyclin A/E‐Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1‐Cdk4 is different from S/T‐P‐X‐K/R, which is the consensus sequence for phosphorylation by cyclin A/E‐Cdk2 using various synthetic peptides as substrates. Cyclin D1‐Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E‐Cdk2 and A‐Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho‐Ser780 in pRB. We confirmed that cyclin D1‐Cdk4, but not cyclin E‐Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle‐dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F‐1 in vivo. Our data show that cyclin D1‐Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.

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Section: Methodsmentioning

confidence: 99%

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

et al. 1996

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“…Whole-cell lysates (300 mg) were incubated with 5 ml of anti-CDK4 antibody or preimmune mouse IgG in 300 ml of IP buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 0.1% Tween-20, 10% glycerol) at 41C for 1 h. Immune complexes were collected on protein A/G-sepharose beads, washed three times in 0.5 Â IP buffer, and resuspended in 25 ml of kinase buffer containing 20 mm Tris-HCl (pH 7.4), 10 mm MgCl 2 , 1 mm EGTA, 4.5 mm 2-mercaptoethanol, 50 mm ATP, and 15 mCi [g-32 P]ATP in the presence of pRB-GST fusion protein (nt 385-928) (Saijo et al, 1994). After…”

Section: Immunoprecipitation and Rb Kinase Assaymentioning

confidence: 99%

Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c

et al. 2003

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ARG is a tyrosine kinase closely related to ABL, which is oncogenic when fused to the transcriptional repressor ETV6 (ETS translocation variant 6). In this study, we investigated the growth-inhibitory effect of STI571 (signal transduction inhibitor number 571) on ETV6/ARGexpressing cells and its molecular mechanisms using HT93A, a cell line derived from a patient with AML-M3 carrying t(1;12). STI571 effectively suppressed overall tyrosyl phosphorylation of intracellular proteins including ETV6/ARG fusion protein, as well as the growth of HT93A cells with an IC 50 of 200 nm. The growth inhibition was primarily because of cell cycle arrest at G1 phase when cells were treated with 100 nm STI571 for 48 h, and apoptosis was induced after longer exposure (72 h) or by a higher dose (1000 nm). STI571 increased the amount of p18/INK4c after 2 h of culture, when the cell cycle pattern was not yet affected, but not that of other CDK inhibitors (CKI). p18/INK4c was more abundant in G1-enriched fractions than in S-and G2/Menriched fractions of STI571-treated HT93A cells, suggesting that the upregulation of p18/INK4c expression correlates with the cell cycle arrest. Treatment of HT93A cells with antisense oligonucleotides against the Ink4c gene abrogated the growth inhibition by STI571. These results suggest that leukemogenesis by an aberrant ARG kinase involves the suppression of p18/INK4c, which is ubiquitously expressed and considered the major CKI in hematopoietic stem cells. STI571 can be an effective drug for the treatment of leukemias with deregulated ARG kinase activity.

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Interaction of Rat Cdc37-Related Protein with Retinoblastoma Gene Product

Ozaki

Sato²

1996

DNA and Cell Biology

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By using in vitro binding assays and the yeast two-hybrid system, we have found that a full-length rat Cdc37-related protein (RCdc37) could associate specifically with the retinoblastoma susceptibility gene product (pRB). A series of GST-RCdc37 deletion mutants was constructed to define the amino acid sequence required for the interaction with pRB. A GST-RCdc37 (-20 approximately 229) possessed an activity to associate with pRB, whereas GST-RCdc37 (-20 approximately 165) lost its activity, indicating that the amino acid sequence between 166 and 229 of RCdc37 was essential for the association with pRB. Interestingly, there exists a highly conserved pRB-binding motif (LXCXE; X = any amino acid) that is essential for pRB binding of SV40 large T antigen, E1A, and E7 proteins. A similar experiment using a pRB deletion mutant revealed that the carboxy-terminal portion of pRB was required for binding to RCdc37.

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Inactivation of oncoprotein binding by a single Cys⁷⁰⁶‐to‐Tyr substitution in the retinoblastoma protein

Cited by 4 publications

References 29 publications

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c

Interaction of Rat Cdc37-Related Protein with Retinoblastoma Gene Product

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Inactivation of oncoprotein binding by a single Cys706‐to‐Tyr substitution in the retinoblastoma protein

Cited by 4 publications

References 29 publications

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c

Interaction of Rat Cdc37-Related Protein with Retinoblastoma Gene Product

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Inactivation of oncoprotein binding by a single Cys⁷⁰⁶‐to‐Tyr substitution in the retinoblastoma protein