Spy1 interacts with p27Kip1 to allow G1/S progression

Abstract

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.

Figure 1.

Schematic representation of p27. Full-length p27 contains a cyclin-binding region (aa 25–51), a CDK-binding region (aa 53–93), and a nuclear localization signal (NLS; aa 152–166). The two clones isolated from the yeast two-hybrid screen encompassed aa 23–128 of p27 (middle) and aa 32–162 of p27 (bottom).

Figure 2.

Spy1 interacts in vivo with the mouse p27 clones isolated from the two-hybrid screen. (A) 293T cells were transiently transfected with the indicated constructs, lysed, immunoprecipitated with α-myc sera, analyzed by 10% SDS-PAGE, and transferred to nitrocellulose membrane. The membrane was then immunoblotted with α-flag sera to detect flag-tagged Spy1 (top panel). As shown in lanes 11 and 12 of the top panel, flag-Spy1 coimmunoprecipitates with myc-p27^23–128 and myc-p27^32–162, respectively. The membrane was then stripped and reprobed with α-myc sera to demonstrate the presence of the myc-tagged p27 constructs (bottom panel). (B) 293T cells were transfected with the indicated constructs, lysed, immunoprecipitated with α-myc sera, and analyzed as above. Myc-p27 ^25–51, which is the cyclin-binding region of p27, did not appear to bind to flag-Spy1 (lane 11). In contrast, myc-p27 ^43–128, which encompasses the CDK-binding region of p27, interacted with flag-Spy1 (lane 12).

Figure 3.

Human p27 interacts with human Spy1 in vitro. (A) In vitro–translated p27 (TNT-p27) was incubated for 2 h with either in vitro–translated pCS3-MT empty vector (TNT-mock) or in vitro–translated myc-tagged Spy1 (TNT-Spy1). The top panel demonstrates the amount of input TNT-Spy1 protein (lane 2), the middle panel demonstrates the input TNT-p27 protein (lanes 1 and 2). Both samples were immunoprecipitated with α-myc sera, and proteins were separated by 10% SDS-PAGE and immunoblotted with p27 sera (bottom panel). (B) In vitro–translated pCS3-MT (TNT-mock), p27 (TNT-p27), myc-Spy1 (TNT-Spy1), or CDK2 (TNT-CDK2) were separated by 10% SDS-PAGE and immunoblotted with α-CDK2 sera as a control. (C) Purified GST-p27 or GST-control protein conjugated to GST beads were incubated in binding buffer with either in vitro–translated (TNT) mock or Spy1 protein (top panel), both of which were previously immunodepleted for any CDK2 protein as an additional control. A GST pull-down assay was performed. Proteins were separated by 12.5% SDS-PAGE and immunoblotted with α-myc sera to detect immunoprecipitation of GST proteins with myc-tagged proteins (bottom panel). α-GST immunoblot is presented to demonstrate the GST proteins present in the GST pull-down experiment. Different exposures of the same immunoblot are presented in the middle panel because of the high amount of protein in the GST-control lanes.

Figure 4.

p27 binds to Spy1 in vivo. (A) 293T cells were transiently transfected with the indicated constructs, lysed, immunoprecipitated with α-p27 sera, analyzed by 10% SDS-PAGE, and transferred to nitrocellulose membrane. The membrane was then immunoblotted with α-myc sera to detect myc-tagged Spy1 (top panel). As shown in lane 8 of the top panel, p27 can coimmunoprecipitate with myc-tagged Spy1. The bottom panel demonstrates the approximately equal expression of exogenous p27. (B) 293T cells were transfected with mock or flag-Spy1 constructs, starved, lysed, and immunoprecipitated with α-p27 sera. Immunoblotting with α-flag sera shows that flag-Spy1 interacts with endogenous p27 (lane 4). (C) 293T cells were starved, lysed, and immunoprecipitated with α-p27 or α-Spy1 sera. Immunoblotting demonstrates that endogenous Spy1 interacts with endogenous p27 (lanes 1 and 2). A sample with no primary antibody was used as a negative control (lane 3).

Figure 5.

p27 and Spy1 colocalize in the nucleus. COS-1 cells were transiently transfected with p27, myc-Spy1, or p27 plus myc-Spy1. (A) p27 localizes to the nucleus as shown in panel a. Similarly, myc-Spy1 localizes in the nucleus as shown in panel c. (B) Cells transfected with both p27 and myc-Spy1 demonstrate that both proteins localize to the nucleus (panel c). Hoechst dye was used to stain the nucleus.

Figure 6.

p27 interacts with Spy1 and CDK2. 293T cells were transfected with the indicated constructs. The lysates were immunoprecipitated with α-p27 or α–CDK2 sera and immunoblotted for Spy1, CDK2, and p27. Lane 8 demonstrates that flag-Spy1 (top panel), endogenous CDK2 (middle panel), and p27 (bottom panel) can coimmunoprecipitate. The top panel of lane 11 shows that Spy1 interacts with CDK2 and that this interaction is increased when p27 is overexpressed as demonstrated in lane 12.

Figure 7.

Spy1 can overcome a p27-induced G₁ arrest. (A) NIH3T3 wild-type cells were transfected with the indicated constructs and treated with BrdU for 2 h. The cells were then fixed and examined by immunofluorescence for BrdU incorporation by examination of GFP-positive cells. The graphed data indicate that there is an increase in DNA synthesis when p27 and Spy1 are coexpressed in comparison to p27 alone. (B) The lysates from the BrdU experiment were analyzed by 10% SDS-PAGE; immunoblotting with α-flag, α-GFP, and α-p27 sera show approximately equivalent levels of protein expression.

Figure 8.

Spy1 enhances histone H1 kinase activity in the presence of p27. (A) 293T cells were transfected with the indicated constructs. The lysates were immunoprecipitated with α-CDK2 sera and subjected to an in vitro kinase assay. Lane 2 shows that p27-transfected cells have decreased kinase activity, but Spy1-transfected cells exhibit kinase activity higher than mock-transfected cells (lane 3). When both Spy1 and p27 are cotransfected, CDK2 histone H1 kinase activity is increased as shown in lane 4 compared with p27 alone. (B) The lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with flag-Spy1, CDK2, and p27 antisera to demonstrate equivalent protein expression.

Figure 9.

Spy1-enhanced proliferation is dependent on endogenous p27. (A) MEF–/– cells were transfected with the indicated constructs, medium was changed 24 h posttransfection, and cells were counted via trypan blue exclusion 48 h posttransfection. The graphed data indicate that exogenous p27 slows proliferation in the MEF–/– cells and that Spy1 can overcome this inhibition of proliferation. Spy1 does not enhance proliferation over mock control in cells lacking endogenous p27. The data are one representative experiment of four. Error bars indicate the SEM of three separate transfections. (B) Lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with α-myc-Spy1, and α-p27 sera to demonstrate protein expression. (C) Endogenous Spy1 was immunoprecipitated from either NIH3T3 cells null for p27 (3T3–/–) or NIH3T3 wt (3T3wt). Both lysate and immunoprecipitated samples were separated by 10% SDS-PAGE and immunoblotted with α-CDK2 sera (top panel) or α-p27 sera (bottom panel). (D) Endogenous CDK2 was immunoprecipitated from both 3T3wt and 3T3–/– cell types. Lysate samples and the immunoprecipitated samples were analyzed by 10% SDS-PAGE, and immunoblotting was carried out with α-Spy1 sera.