Cell cycle-dependent expression of SEI-1 and its influence over p16^INK4a activity. (A) Early passage (35 PDL) TIG-3 cells were rendered quiescent in medium containing 0.2% serum for 4 days, then stimulated to proliferate by addition of 20% FCS. Total cell lysates were prepared at various time points and 150 μg of protein were subjected to Western blotting with antibodies indicated at right. (B) HPLC-purified cyclin D1–CDK4 complexes, prepared from baculovirus-infected Sf9 cells, were preincubated with His–p16^INK4a, with or without His–p34^SEI-1 or His–Id1 as indicated, and then assayed for kinase activity toward GST–Rb. (C) ³⁵S-Labeled in vitro-translated p16^INK4a, CDK4, CDK6, and p53 proteins (top) were mixed with 10 ng of His–p34^SEI-1 protein (bottom). The samples were analyzed directly by SDS-PAGE (top) or after immunoprecipitation with an antibody against p34^SEI-1 (bottom).

Protection of cyclin D1–CDK4 complex from p16^INK4a by p34^SEI-1. Both in vitro-translated ³⁵S-labeled Myc-tagged cyclin D1 and CDK4 (A) or unlabeled CDK6 (B) were mixed with His–p16^INK4a, p34^SEI-1, or Id1 as indicated. The mixtures were immunoprecipitated with antisera to CDK4 (A) or CDK6 (B) and analyzed by SDS-PAGE. Labeled D1 proteins were detected by autoradiography. (B) (C) In vitro-translated ³⁵S-labeled Myc-tagged-cyclin D1 and Cdk4 were mixed with the proteins as indicated, immunoprecipitated with anti-p16 antibody, and analyzed by SDS-PAGE. Labeled proteins were detected by autoradiography (top). These immunoprecipitated samples were also subjected to Western blotting with anti-His-tag antibody (bottom).

Effects of p34^SEI-1 on cyclin D1–CDK4 complex formation in vivo. (A) Early passage TIG-3 cells were infected with retrovirus that encodes SEI-1 (lane 2) or empty vector (lane 1) and selected by puromycin. Selected cells were pooled and were incubated with a medium containing 1% serum for 4 days, then lysates were prepared and immunoprecipitated with anti-cyclin D1 antibody and immunoblotted with either anti-CDK4 (middle) or anti-cyclin D1 (bottom) antibody. Expression level of p34^SEI-1 was confirmed by Western blot with anti-SEI-1 antiserum (top). (B,C,D,E) Early passage TIG-3 cells were cultured in a low serum (0.2% FCS) medium for 3 days, then treated with either antisense oligomer against the SEI-1 gene or a control oligomer for 24 hr. (B) Total RNAs were prepared from both control (lane 1) and antisense oligomer (lane 2)-treated cells that had been incubated with high-serum (20% FCS) medium for 2 hr and were subjected to Northern blotting with either SEI-1 (top) or β-actin (bottom) cDNA probes. (C) Cell lysate were prepared from both control (lanes 1,2) and antisense oligomer (lane 3)-treated cells incubated with high serum medium for 2 hr in the presence of [³⁵S]methionine. Cell lysates were then immunoprecipitated with antisera to SEI-1 in the absence (lanes 2,3) or presence (lane 1) of competing SEI-1 peptide and analyzed by SDS-PAGE. (D) Cell lysates were prepared from both control (lane 2) and antisense oligomer (lane 3)-treated cells that were incubated with high serum medium for 8 hr and were immunoprecipitated with anti-cyclin D1 antibody and immunoblotted with either anti-CDK4 (top) or anti-cyclin D1 (bottom) antibody. 1/100 volume of total cell lysate from TIG-3 cells was directly immunoblotted as a positive control (lane 1). (E) Cells were incubated with high-serum medium for 20 hr in the presence of [³H]thymidine, then fixed and developed. The percentages of [³H]thymidine-incorporated cells were measured in both control (lane 1) and antisense oligomer (lane 2) treated cells.

Interaction of p34^SEI-1 with CDK4. (A) Schematic representation of p34^SEI-1 and its interaction activity. (B) A series of truncation mutants of ³⁵S-labeled p34^SEI-1 proteins (top) were incubated with His–CDK4 protein. Bound ³⁵S-labeled p34^SEI-1 proteins were detected by autoradiography (bottom). (C) Schematic representation of CDK4 and its interaction activity. (D) A series of truncation mutants of ³⁵S-labeled CDK4 proteins (top) were incubated with either His–p16^INK4a (middle) or His–p34^SEI-1 protein (bottom). Bound ³⁵S-labeled CDK4 proteins were detected by autoradiography (middle, bottom). (E) Cos7 cells were transfected with expression vectors for Flag-tagged p34^SEI-1 and/or CDK4 as indicated (top) and collected 48 hr later. Specific interactions were analyzed by immunoblotting with antibodies indicated at left after immunorecipitation with antibodies indicated at bottom (lanes 1–5). (F) p34^SEI-1 immunoprecipitates prepared from C33A cells in the absence (lane 2) or presence (lane 3) of competing SEI-1 peptide were subjected to Western blotting with anti-CDK4 antibody. 1/100 volume of total cell lysate from C33A cells was directly immunoblotted as a postivie control (lane 1).

Effects of p34^SEI-1 on cell growth. (A) Clones of rat 3Y1 fibroblasts stably transfected with a p34^SEI-1 expression vector (F-2 and F-11) or empty vector (N-2) were monitored for p34^SEI-1 expression by immunoblotting with an antiserum (TH1) against the carboxyl terminus of p34^SEI-1. (B) The clones were placed in either 10% serum (left), 3% serum (middle), or 1% serum (right), and relative cell numbers were compared at 12-hr intervals. (□) N-2; (●) F-2; (○) F-11. (C) N-2 and F-11 cells were maintained in medium containing 1% serum for 4 days. Cell lysates were then immunoprecipitated with either CDK4 antiserum or NRS. The precipitates were tested for their ability to phosphorylate GST–Rb protein (top) and were also subjected to Western blotting with anti-CDK4 antibody (bottom). (D) 3Y1 cells transfected with expression plasmids encoding wild-type (WT) or CDK4-binding-deficient mutant; amino acids 134–236 (Mut) of p34^SEI-1 or empty vector (Vec), pcDNA3 (Invitogen), were selected against G418 for 2 weeks and pooled. This pooled population was monitored for p34^SEI-1 expression by immunoblotting with an antiserum (TH1). (NS) Nonspecific association to TH1 antiserum in 3Y1 cell lysate. (E) The pooled cells were placed in the medium containing 1% serum and relative cell numbers were compared at 12-hr intervals. (□) Vec; (●) Mut; (○) Wt.