Pim-1 binds to Skp2. A, HEK293T cells were transfected with the indicated plasmids, protein immunoprecipitated (IP) with HA antibody and immunoblotted with FLAG or Pim-1 antibody. Lysates of cells used for this assay are probed with identical antibodies. B, HEK293T cells were serum-starved (0.2%) for 48 h prior to the addition of 15% FBS at 0 time. Cells were then harvested at the indicated time points, and coimmunprecipitation (co-IP) was performed. C, GST-Skp2 proteins were incubated overnight with His-tagged Pim-1 or Ubc3 proteins purified from E. coli at 4 °C, washed with PBS, and subjected to immunoblot analysis (upper panel). GST and GST-Skp2 were stained with Coomassie Brilliant Blue (lower panel). D, HEK293T cells were transfected with the indicated plasmids, treated with 10 μm MG132 for 6 h, and ubiquitination as measured by binding to nickel-nitrilotriacetic acid (Ni-NTA) beads (see “Experimental Procedures”) followed by an immunoblot with anti-HA antibody. Immunoblot (IB) analysis was performed on total cell lysates from these HEK293T cells (two lower panels).

Pim-1 kinase phosphorylates Cdh1 and impairs its binding to CDC27. A, HEK293T cells were transfected with HA-Cdh-1, Pim-1, or kinase-dead Pim-1 K67M or NT81, immunoprecipitated (IP) with HA antibody followed by Western blotting with antibodies to CDC27 and Pim-1. Lysates from these cells were immunoblotted (IB) with antibody as shown. B, HeLa cells were cotransfected with HA-Cdh1 and Pim-1 or scrambled siRNA plasmids before harvesting for coimmunoprecipitation analysis. Levels of transfected proteins in lysates were monitored by immunoblotting. C, HeLa cells were transfected with the indicated shRNA plasmids followed by a double-thymidine block. After release from the block cell, lysates were prepared at 8 and 16 h and subjected to immunoblot analysis. The arrows indicate phosphorylated and unphosphorylated Cdh-1. D, in vitro translated HA-tagged Cdh1 or Cdc20 was incubated with recombinant Pim-1 and [γ-³²P]ATP in an in vitro kinase assay. Autoradiography (upper panel) and immunoblot (lower panel) analyses were performed. The phosphorylation of Cdh1 or Cdc20 was quantified by densitometry from three independent experiments after normalization to the loaded protein. E, HeLa cells were transfected with HA-Cdh1 and human Pim-1, and the kinase inhibitors roscovitine (20 μm) and SMI-4a (10 μm) were added 1 h before labeling with ³²P_i. HA-Cdh-1 was immunoprecipitated, and autoradiography (top panel) and immunoblot analysis were performed (second panel). The phosphorylation of Cdh1 was quantified by densitometry from three independent experiments with normalization to Cdh1 expression (HA). A coimmunoprecipitation experiment was performed to monitor Cdh1 and CDC27 interaction under the same experimental conditions (lower three panels). F, HEK293T cells were transfected with FLAG-Skp2, myc-Cdh-1, and HA-tagged WT and kinase-dead (K67M) Pim kinase, and the extracts were immunoblotted with the specified antibodies. G, HeLa cells were transiently transfected with a siRNA to Pim-1 or a scrambled sequence. Forty-eight h after transfection, extracts of these cells were probed on Western blots with the listed antibodies (left two lanes). HeLa cells were treated with 20 μm Pim kinase inhibitor SMI-4a or K00135 for 16 h, extracts were prepared, and immunoblotting was carried out with the identified antibodies (right three lanes).

Model of Pim-1 regulation on Skp2 degradation. Nonphosphorylated Skp2 binds to E3 ligase APC/C^Cdh1 and gets ubiquitinated (Ub) followed by proteasome-mediated degradation. Pim-1 kinase phosphorylates Skp2 on multiple sites: Ser⁶⁴, Ser⁷², and Thr⁴¹⁷. Furthermore, the phosphorylation of Cdh1 by Pim-1 reduces Cdh1 and APC/C interaction. Both Pim-1 actions result in decreased Skp2 ubiquitination and consequently increased Skp2 stability.

Regulation of Skp2 protein levels by Pim-1. A, HeLa cells were transiently transfected with cDNAs encoding green fluorescent protein (GFP), Pim-1, kinase-dead Pim-1 (K67M), or a siRNA to Pim-1 together with GFP, or Skp2, or Pim-1, or a scrambled sequence. Forty-eight h after transfection, extracts of these cells were probed on Western blots with the listed antibodies. B, HeLa cells were treated with various concentrations of Pim kinase inhibitor SMI-4a for 16 h, extracts were prepared, and immunoblotting was carried out with the identified antibodies. C, HeLa cells were transfected with the indicated siRNA plasmids followed by a double-thymidine block treatment (see “Experimental Procedures”). Lysates were prepared at the indicated time points after release from the thymidine block and subjected to immunoblot analysis. Arrows indicate phosphorylated and unphosphorylated forms of Cdh1. D, mouse prostate epithelial cells (MPECs) stably transfected with a control vector (pLNCX) or a human Pim-1-expressing plasmid were treated with HGF (50 ng/ml) for 24 h followed by immunoblot analysis. E, 24 h after transfection with expression plasmids (time 0), HEK293T cells were incubated for the indicated times with cycloheximide (CHX, 100 μg/ml) followed by immunoblot analysis with FLAG or β-actin antibodies. Densitometric analysis was performed using ImageJ software to quantify the expression of Skp2. Skp2 band intensity was normalized to β-actin, then normalized to the t = 0 controls.

Pim-1 phosphorylates Skp2. A, FLAG-Skp2 was immunoprecipitated from HEK293T cells, incubated with recombinant His-tagged Pim-1 for 30 min with or without SMI-4a for an in vitro kinase assay (“Experimental Procedures”) followed by SDS-PAGE autoradiography (upper panel) and immunoblot analyses (lower panel). The phosphorylation of FLAG-Skp2 was quantified by densitometry from three independent experiments after normalization to input. B, C-terminal sequence of Skp2 contains a Pim-1 consensus site. C, GST-tagged Skp2 proteins or a T417A mutant was incubated with recombinant GST-Pim-1 and [γ-³²P]ATP for 30 min, and subjected to SDS-PAGE followed by autoradiography. The phosphorylation of GST-Skp2 was quantified by densitometry from three independent experiments with normalization to Coomassie Blue staining. D, wild type (WT) FLAG-Skp2 and its mutants T417A, S64A, S72A, and S64A/S72A were immunoprecipitated from HEK293T cells, incubated with recombinant GST-tagged Pim-1 and [γ-³²P]ATP for 30 min, followed by SDS-PAGE autoradiography (upper panel) and immunoblot analysis (lower panel). The phosphorylation of FLAG-Skp2 was quantified by densitometry from three independent experiments following normalization to the level of protein input. E, HeLa cells were pretreated with roscovitine (20 μm), wortmannin (1 μm), or SMI-4a (10 μm) for 1 h, transfected with human Pim-1 and Skp2, and labeled with ³²P_i followed by FLAG immunoprecipitation, autoradiography (upper panel), and FLAG/Pim-1 immunoblots (two lower panels). The phosphorylation of FLAG-Skp2 was quantified by densitometry from three independent experiments along with normalization to Skp2 expression. F, HeLa cells were transfected with the indicated Skp2 constructs and synchronized in M phase by mitotic shake-off of cells obtained after release from a thymidine-nocodazole block. The cells were then replated and allowed to progress through the cell cycle in the presence of cycloheximide (100 μg/ml). Immunoblot analysis was performed at specific time points using antibodies to cyclin B and phosphohistone H3 Ser¹⁰ (p-H3 (S10)) as controls. Densitometric analysis was performed using ImageJ software to quantify the expression of Skp2. Skp2 band intensity was normalized to β-actin and then normalized to the t = 0 controls. G, HEK293T cells were transfected with a FLAG-tagged p27 Skp2 construct or a GFP control. Expression of exogenous p27 and Skp2 is measured by immunoblotting.

Pim-1 is required for Skp2 to signal cell cycle S phase entry. A, HeLa cells were treated with a double-thymidine block and released into fresh medium. Cells were then harvested at the indicated time points and subjected to FACS (left panel) and immunoblot analysis (right panel). The arrow denotes the Pim-1 signal. * indicates a nonspecific signal. B, Rat1 cells were transduced with a lentivirus carrying the indicated cDNAs. Cells were maintained in low serum conditions (0.2%) for 48 h before harvested for FACS (upper panel) and immunoblot (lower panel) analyses. Percent of S phase cells was compared with vector control, except where indicated by a bracket. C, the experiment was performed as in B except that SMI-4a (5 μm) was added 3 h before a 20% FBS stimulation (16 h). S phase induction was also determined by BrdU incorporation assay. Brackets indicate comparison of with and without SMI-4a treatment.