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1.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 6. hKIS kinase activity is growth factor-dependent and regulates p27Kip1 in vivo. hKIS interacts with endogenous p27Kip1. Fibroblasts from p27Kip1 wild-type (p27+/+) and null (p27–/–) mice were incubated with AMP-PNP, p27Kip1 was immunoprecipitated using p27Kip1 C19 antibody, and a western blot was performed (lanes 2 and 3). hKIS and p27Kip1 protein levels in p27+/+ and p27–/– cells were determined using a western blot (lanes 4 and 5). To demonstrate a reciprocal p27Kip1 and hKIS interaction, hKIS was immunoprecipitated with hKIS 291 antibodies, and a western blot was performed with p27Kip1 K25020 antibody (lanes 6 and 7).

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
2.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 4. hKIS phosphorylates p27Kip1 in vivo. HEK 293 cells transiently expressing HA-tagged wild-type p27Kip1, hKIS, an hKIS(K54A) mutant or a p27(S10A) mutant were metabolically labelled with [32P]orthophosphate. HA-tagged p27Kip1 and p27(S10A) proteins were immunoprecipitated with an HA antibody, subjected to SDS–PAGE and analysed by autoradiography. (Lane 1) a control immunoprecipitation using IgG; (lane 2) wild-type p27Kip1; (lane 3) expression of wild-type p27Kip1 and hKIS; (lane 4) expression of p27Kip1(S10A) and hKIS; (lane 5) expression of wild-type p27Kip1 and hKIS(K54A). A decrease in p27Kip1 protein might be expected following expression with hKIS (lane 3), but was not observed as the ratio of hKIS to p27Kip1 was 3:1.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
3.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 8. hKIS stabilizes p27Kip1 in G1 cells. (A) NIH 3T3 cells were serum starved for 24 h and transfected with wild-type p27Kip1, p27(S10A), p27(S10D), or p27Kip1 and hKIS. The cells were pulse- labelled for 2 h with [35S]methionine, and chased for the indicated time points in media containing 20% FBS. Cell lysates were immunoprecipitated with p27Kip1 C19 antibodies, and the labelled p27Kip1 protein was analysed by autoradiography. (B) Densitometric analysis of p27Kip1 degradation rate. The intensity of the bands in (A) is expressed as a percentage of the time point t0. Data are from an experiment that was repeated twice with similar results.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
4.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 1. Identification of hKIS and interaction with p27Kip1. (A) hKIS interacts with the QT domain of p27Kip1 in a yeast two-hybrid assay. hKIS was cotransfected into yeast with either p27Kip1, p27Kip1 [1–88 amino acids (aa)], p27Kip1 (144–198 aa), p57Kip2, p57Kip2 (1–89 aa), p57Kip2 (260–335 aa) or p21Cip1. β-galactosidase assay on selection plates was performed and the intensity of staining was categorized. CDK CS, cyclin-dependent kinase consensus sequence; NLS, nuclear localization signal; PCNA, proliferating cell nuclear antigen. +++, very strong; ++, strong; (+/–), weak; –, no staining. (B) Interaction between hKIS and CKIs. hKIS is coimmunoprecipated with p27Kip1, p16Ink4, p21Cip1 or p57Kip2. GST indicates pGEX-6P backbone control. Input is [35S]methionine-labelled hKIS control (10% of the total amount). Data are from an experiment that was repeated twice with similar results.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
5.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 2. Phosphorylation of CKIs by hKIS. (A) hKIS phosphorylates p27Kip1. In vitro-transcribed and -translated hKIS was immunoprecipitated with Anti-Xpress antibody and incubated with GST- purified p27Kip1. (Lane 1) [35S]methionine-labelled hKIS control; (lane 2) immunoprecipitated pcDNA 3.1 Anti-Xpress tag backbone incubated with p27Kip1 as a negative control; (lane 3) immunoprecipitated hKIS(K54A) mutant incubated with p27Kip1; (lane 4) immunoprecipitated hKIS incubated with p27Kip1. (B) hKIS phosphorylates p27Kip1 at the N-terminal domain. N-terminal (codons 1–88) or C-terminal (codons 144–198) fragments of p27Kip1 were purified as GST fusion proteins, and a kinase assay with hKIS was performed (upper panel). An equal amount of the GST fusion protein (40 pmol) was used in the kinase assay (upper panel), as stained by Coomassie Brilliant Blue (lower panel). (C) hKIS phosphorylates p27Kip1 on S10. Serine 10 was substituted with alanine in a GST fusion protein p27Kip1(S10A). The kinase assay was performed with hKIS (+, lanes 2 and 4) or a kinase-dead mutant, hKIS(K54A) (–, lanes 1 and 3).

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
6.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 10. hKIS promotes cell cycle progression. (A) hKIS releases G1 arrest in p27Kip1 cells. The vectors were expressed in HEK 293 cells with a CD2 reporter and analysed by propidium iodine staining of CD2-positive cells by FACS after 48 h. Experiments were performed in triplicate. Data are represented as means ± SEM. (B and C) hKIS downregulates p27Kip1 protein levels, leading to cell cycle progression. The indicated vectors were expressed in HEK 293 cells, and cell lysates were analysed by western blotting using p27Kip1 K25020 antibody for detection of p27Kip1 and p27(S10A) (B, upper panel), a mouse anti-Flag antibody for hKIS and hKIS(K54A) (B, lower panel), and p21Cip1 C19 antibodies for p21Cip1 (C). Protein levels were measured by densitometry using an image analysis system (data not shown). Data are presented from an experiment that was repeated twice with similar results.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
7.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 5. hKIS is a growth factor-dependent kinase and localizes to the nucleus. (A) Specificity of polyclonal hKIS 291 antibodies. The antibodies were absorbed with GST fusion proteins, and immunoblotting was performed using NIH 3T3 lysates. (Lane 1) GST alone; (lane 2) hKIS blocked with GST–hKIS protein; (lane 3) an irrelevant GST fusion protein, GST–C19. (B) A mouse monoclonal 3H2 antibody recognizes hKIS. An immunoblot was performed using NIH 3T3 lysates after incubation of the mouse hKIS antibody with GST fusion proteins. (Lane 1) hKIS blocked with GST–hKIS protein; (lane 2) GST alone. (C) hKIS localizes to the nucleus. NIH 3T3 cells were serum starved for 36 h (0.1% FBS, upper panel), and then cells were serum stimulated for 6 h in 10% FBS (lower panel). Immunofluorescence and confocal microscopy were performed using hKIS 291 antibodies (left panel), a p27Kip1 antibody K25020 (second panel from left) or both antibodies (third panel from left). A nuclear stain, DAPI, is shown on the far right panel.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
8.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 7. hKIS kinase activity is associated with S10 phosphorylation of endogenous p27Kip1. (A) KIS kinase activity increases following serum induction. NIH 3T3 cells were serum starved for 36 h, followed by serum stimulation for 0–8 h. hKIS protein was immunoprecipitated with hKIS 291 antibodies, and a kinase assay was performed with recombinant p27Kip1 as a substrate (lanes 1–4, upper panel). hKIS protein levels were visualized by western blot analysis (lanes 5–7, lower panel). (B) Quantification of phosphorylated p27Kip1 in NIH 3T3 cells treated with lactacystin. The relative intensity of phosphorylated recombinant p27Kip1 by hKIS was determined at the indicated time points by densitometry (left). Cells were lysed at the indicated time points. Data are from an experiment that was repeated twice with similar results. (C) S10 is a major phosphorylation site following serum stimulation. Two-dimensional tryptic phosphopeptide mapping was performed on NIH 3T3 cells serum starved for 36 h, followed by serum stimulation for 0–8 h. Phosphopeptides containing S10 are indicated by arrows. An asterisk indicates the origin of migration, and arrows show the directions of separation by TLC and electrophoresis.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
9.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 9. hKIS phosphorylation on S10 causes nuclear export of p27Kip1. (A) Subcellular localization of endogenous p27Kip1 in NIH 3T3 cells transfected with hKIS. Cells were serum starved for 24 h, then serum stimulated for 8 h in the absence (–) or presence (+) of leptomycin B (LMB) 2 ng/ml, stained with p27Kip1 or hKIS antibodies, and examined by confocal microscopy. In serum-starved cells not expressing KIS, endogenous p27Kip1 is nuclear. Serum stimulation leads to redistribution to the cytoplasm. In contrast, in serum-starved cells expressing KIS, endogenous p27Kip1 is nuclear and cytoplasmic. After serum stimulation, endogenous 27Kip1 is cytoplasmic in these cells. The arrows indicate endogenous p27Kip1 in cells expressing KIS (upper panel) and transfected KIS (middle panel). A nuclear DAPI stain is shown in the lower panel. (B) Quantitative analysis of the cellular localization of endogenous p27Kip1 in cells transfected with hKIS. Results are expressed as the percentage of cells demonstrating both cytoplasmic and nuclear staining. Data are expressed as means ± SEM of three experiments. (C) Nuclear export precedes Cdk2 activation. Cells were serum starved for 24 h and stimulated with serum for the indicated times. p27Kip1 expression was examined by immunoblotting with p27Kip1 antibodies. Cdk2 activity was assayed using histone H1 as substrate.

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
10.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 11. hKIS is required for S10 phosphorylation. (A) Reduced hKIS in HEK 293 cells transfected with hKIS siRNA but not control siRNA. Cells were transfected with hKIS siRNA or control (Co) siRNA. At the indicated time points, cells were harvested and cell lysates were generated. Samples were immunoblotted with antibodies to hKIS. (B) Depletion of hKIS causes an absence of S10 phosphorylation. Cells were transfected as in (A). Six hours prior to lysis, cells were treated with lactacystin. Cells were harvested 30 h following transfection, and cell lysates were immunoblotted with a monoclonal p27 S10-p antibody (upper panel) or a monoclonal p27Kip1 antibody (lower panel). (C) hKIS is required for nuclear export of p27Kip1. Cells were transfected with KIS siRNA or Co siRNA, harvested at 0, 24 or 48 h, and immunoblotting of cell lysates was performed with antibodies to KIS (upper left panel). Immunofluorescence for p27Kip1 was also performed, and the number of cells expressing cytoplasmic and nuclear p27Kip1 at 0, 24 and 48 h was counted (lower left panel). A minimum of 200 cells were scored. The results are expressed as the percentage of cells demonstrating both cytoplasmic and nuclear staining. Cells treated with Co or KIS siRNA and Co siRNA were immunostained for KIS and p27Kip1 + KIS 48 h after transfection, and examined by confocal microscopy (right panel). (D) Depletion of hKIS leads to G1 arrest. Cells were transfected with Co or KIS siRNA, harvested at 30 h, and a FACS analysis was performed. (E) p27Kip1 is a critical target for KIS. p27+/+ and p27–/– fibroblasts were treated with Co or KIS siRNA and harvested 48 h later. Cell lysates were immunoblotted with KIS antibodies (upper panel) and FACS analysis was performed (lower panel).

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.
11.

Figure. From: A growth factor-dependent nuclear kinase phosphorylates p27Kip1 and regulates cell cycle progression.

Fig. 3. hKIS phosphorylates p27Kip1 on S10. (A) Two-dimensional tryptic phosphopeptide mapping of wild-type and mutant p27Kip1. p27Kip1, p27(S10A) or p27(T187A) were expressed in HEK 293 cells and metabolically labelled with [32P]orthophosphate in the presence of 50 µM lactacystin. The recombinant proteins were immunoprecipitated with p27Kip1 C19 antibody and subjected to 2D tryptic phosphopeptide mapping. Major phosphopeptides are numbered 1 and 2. Phosphopeptides containing S10 are indicated by arrows. An asterisk indicates the origin of migration, and arrows show the directions of separation by thin layer chromatography (TLC) and electrophoresis. (B) Mouse polyclonal antibodies recognize phosphorylated p27Kip1 on S10. A western blot of recombinant p27Kip1 phosphorylated by hKIS was performed with a p27S10-p antibody absorbed with non- phosphorylated recombinant p27Kip1 protein (Control, lane 2) or S10-p p27Kip1 peptide (lane 1). Phosphorylated p27Kip1 was incubated with (+) or without (–) CIAP, and a western blot with p27S10-p antibody was performed (lanes 3 and 4). The same blot was stripped and reprobed with a monoclonal p27Kip1 K25020 antibody (lanes 5 and 6). Recombinant p27Kip1 was phosphorylated by hKIS in the presence of [γ-32P]ATP incubated with (+, lane 8) or without CIAP (–, lane 7). HEK 293 cell lysates were analysed on SDS–PAGE and a western blot was performed using the p27S10-p antibody (upper panel, lanes 9 and 10). The same blot was stripped and reprobed with a p27Kip1 C19 antibody (lower panel, lanes 9 and 10). (C) hKIS phosphorylates p27Kip1 on S10. Recombinant p27Kip1 protein was incubated with immunoprecipitated, in vitro-transcribed and -translated hKIS (+, lane 2), and a western blot with p27S10-p antibody was performed. The same blot was stripped and reprobed with a p27Kip1 K25020 antibody (lanes 1 and 2, lower panel).

Manfred Boehm, et al. EMBO J. 2002 July 1;21(13):3390-3401.

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