Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors - PubMed (original) (raw)

Cyclin D-CDK subunit arrangement is dependent on the availability of competing INK4 and p21 class inhibitors

D Parry et al. Mol Cell Biol. 1999 Mar.

Abstract

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G0-G1-S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Assembly of active cyclin D-CDK complexes in response to mitogenic signals is negatively regulated by INK4 family members. Here we show that although all four INK4 proteins associate with CDK4 and CDK6 in vitro, only p16(INK4a) can form stable, binary complexes with both CDK4 and CDK6 in proliferating cells. The other INK4 family members form stable complexes with CDK6 but associate only transiently with CDK4. Conversely, CDK4 stably associates with both p21(CIP1) and p27(KIP1) in cyclin-containing complexes, suggesting that CDK4 is in equilibrium between INK4 and p21(CIP1)- or p27(KIP1)-bound states. In agreement with this hypothesis, overexpression of p21(CIP1) in 293 cells, where CDK4 is bound to p16(INK4a), stimulates the formation of ternary cyclin D-CDK4-p21(CIP1) complexes. These data suggest that members of the p21 family of proteins promote the association of D-type cyclins with CDKs by counteracting the effects of INK4 molecules.

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Figures

FIG. 1

FIG. 1

Gel filtration analysis of CDK4 and CDK6. EL4 whole-cell extract was separated and immunoprecipitated with αCDK6 serum (A) or αCDK4 serum (B) followed by blotting with the precipitating antiserum to reveal the different complexes. Blots were reprobed with αD1 and αD2 sera (C and D). (E) Column fractions were immunoprecipitated with αD2 serum and immunoblotted with a mixture of αCDK4 and αCDK6 sera. This blot was then reprobed with αD2 (F). (G) CDK4 kinase assays on similar EL4 fractions, with GST-RBCT used as a substrate. M, molecular mass markers; IP, immunoprecipitation.

FIG. 2

FIG. 2

INK4-CDK4 and INK4-CDK6 interactions. (A) Cell lysates from EL4, WI38, and CEM cell lines were subjected to immunoprecipitation with specific αINK4 serum with (+) or without (−) peptide block as indicated. Anti-CDK4, αCDK6, and αD2 immunoprecipitations were included as controls. Immune complexes were separated by SDS-PAGE and immunoblotted with a mixture of αCDK4 and αCDK6 sera. Similar analyses were then performed on fractionated whole-cell extracts. (B) Fractionated EL4 extract was immunoprecipitated with αp15INK4b and immunoblotted with αCDK4-αCDK6. (C) Similarly, WI38 lysate was fractionated and immunoprecipitated with αp16INK4a, followed by blotting with αCDK4-αCDK6. Likewise, EL4 and CEM fractions were immunoprecipitated with αp18INK4c (D) and αp19INK4d (E), respectively, and probed with αCDK4-αCDK6. IP, immunoprecipitation.

FIG. 3

FIG. 3

In vitro binding of INK4 inhibitors to CDK4 and CDK6. (A) Freshly prepared D1, CDK2, CDK4, and CDK6 in vitro translates were mixed with individual INK4 in vitro translates, as indicated (A). Complexes were immunoprecipitated using specific antisera, separated by SDS-PAGE in a 12% gel, and visualized by autoradiography. (B) Aliquots of input in vitro translates were separated on another gel as a loading control.

FIG. 4

FIG. 4

Stability of p18INK4c-CDK complexes. Logarithmically growing cultures of EL4 (A) and ML1 (B) cells were pulse-labelled with [35S]methionine and chased with medium containing unlabelled amino acids. At the time points indicated, cell extracts were prepared and subjected to immunoprecipitation (IP) with specific αp18INK4c sera. Immune complexes were separated by SDS-PAGE in a 12% gel.

FIG. 5

FIG. 5

Analysis of p21CIP1 and p27KIP1 by gel filtration. EL4 whole-cell extracts were prepared and fractionated over Superdex 200. (A) Fractions were immunoprecipitated with αp21CIP1 sera and immunoblotted with a mixture of αD1-αD2 and αCDK4 sera (upper panel). The blot was then reprobed with αp21CIP1 (lower panel). (B) Immunoprecipitates from a similar EL4 fractionation using αp27KIP1 sera were immunoblotted in the same way. (C) α-D2 serum was used to immunoprecipitate complexes following gel filtration. After SDS-PAGE, the immune complexes were immunoblotted with sera specific for p21CIP1 (upper panel) or p27KIP1 (lower panel). (D) Mock-depleted and αp21CIP1-αp27KIP1-immunodepleted extracts of EL4 and MCF7 cells were subjected to immunoprecipitation with αD1-D2-D3, αp21CIP1-αp27KIP1 or normal rabbit serum, as indicated. Immune complexes were separated by SDS-PAGE and immunoblotted with a mixture of αD1/D2/D3 sera. IP, immunoprecipitation.

FIG. 6

FIG. 6

Relative stabilities of p16INK4a and αp21CIP1-bound CDK4. Logarithmically growing cultures of WI38 cells were pulse-labelled with [35S]methionine and chased with medium containing unlabelled amino acids. At the time points indicated, extracts were prepared and immunoprecipitated with specific αp16INK4a (A) or αp21CIP1 (B) sera. Immune complexes were separated by SDS-PAGE in a 12% gel. (C) Metabolically labelled CDK4 reimmunoprecipitated from an αp21CIP1 pulse-chase. IP, immunoprecipitation.

FIG. 7

FIG. 7

CDK4 complexes in 293 cells. (A) 293 whole-cell extract was immunoprecipitated with the antisera shown. Immune complexes were separated by SDS-PAGE and immunoblotted with a combination of αCDK4 and αCDK6 sera. Asynchronously growing cultures of 293 cells were pulse-labelled with [35S]methionine and chased with medium containing unlabelled amino acids. At the time points indicated, cell extracts were prepared and immunoprecipitated with specific αp16INK4a (B) or αp18INK4c (C) sera. Immune complexes were separated by SDS-PAGE in a 12% gel. IP, immunoprecipitation.

FIG. 8

FIG. 8

Stimulation of cyclin D-CDK4 interaction by p21CIP1. (A) 293 cells were infected with AdVec or Adp21CIP1 adenoviruses, and lysates were immunoprecipitated with αD2, αp21CIP1, or normal rabbit sera (αNR), as indicated. Following SDS-PAGE, the immune complexes were probed with αD2 sera. (B) Similar infections were immunoprecipitated with αD2, αD3, or normal rabbit sera. Immune complexes were separated by SDS-PAGE alongside equivalents of whole-cell extract prepared from the same samples. Following transfer, the membrane was immunoblotted with αCDK4 serum (upper panel) or αp21CIP1 serum (lower panel). (C) Adp21CIP1 293 cell lysate was separated on Superdex 200. Fractions were collected and immunoprecipitated with αp21CIP1 sera. Immune complexes were separated by SDS-PAGE and immunoblotted with αD2 serum. IP, immunoprecipitation.

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