The essential mitotic peptidyl-prolyl isomerase Pin1 binds and regulates mitosis-specific phosphoproteins

Abstract

Phosphorylation of mitotic proteins on the Ser/Thr-Pro motifs has been shown to play an important role in regulating mitotic progression. Pin1 is a novel essential peptidyl-prolyl isomerase (PPIase) that inhibits entry into mitosis and is also required for proper progression through mitosis, but its substrate(s) and function(s) remain to be determined. Here we report that in both human cells and Xenopus extracts, Pin1 interacts directly with a subset of mitotic phosphoproteins on phosphorylated Ser/Thr-Pro motifs in a phosphorylation-dependent and mitosis-specific manner. Many of these Pin1-binding proteins are also recognized by the monoclonal antibody MPM-2, and they include the important mitotic regulators Cdc25, Myt1, Wee1, Plk1, and Cdc27. The importance of this Pin1 interaction was tested by constructing two Pin1 active site point mutants that fail to bind a phosphorylated Ser/Thr-Pro motif in mitotic phosphoproteins. Wild-type, but not mutant, Pin1 inhibits both mitotic division in Xenopus embryos and entry into mitosis in Xenopus extracts. We have examined the interaction between Pin1 and Cdc25 in detail. Pin1 not only binds the mitotic form of Cdc25 on the phosphorylation sites important for its activity in vitro and in vivo, but it also inhibits its activity, offering one explanation for the ability of Pin1 to inhibit mitotic entry. In a separate paper, we have shown that Pin1 is a phosphorylation-dependent PPIase that can recognize specifically the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. Thus, Pin1 likely acts as a general regulator of mitotic proteins that have been phosphorylated by Cdc2 and other mitotic kinases.

Figure 1

Pin1 levels are constant during the cell cycle. HeLa cells were synchronized at the G₁/S boundary by double thymidine and aphidicolin block and released to enter the cell cycle. To block cells at mitosis, nocodazole (Noc, 50 ng/ml) was added to cells at 8 hr after the release and incubated for another 4 hr (12 Noc) or 6 hr (14 Noc). To obtain pure G₁ cells, mitosis-arrested cells (14 Noc) were plated in a nocodazole-free media for another 4 hr (18 Noc⁻). Cells were harvested at the indicated times and aliquots were subjected to flow cytometric analysis to determine the cell cycle status; the remaining cells were lysed in RIPA buffer. The same amount of total proteins were separated on a SDS-containing gel, transferred to a membrane that was cut into three pieces and probed with anti-cyclin B, anti-Cdc2, and anti-Pin1 antibodies, respectively. Cdc2 levels were similar in all lanes (data not shown).(A) Flow cytometric analysis of the DNA content; (B) immunoblot analysis of cyclin band Pin1 protein levels.

Figure 2

Pin1 interacts directly with a subset of conserved mitosis-specific phosphoproteins both in vitro and in vivo. (A) Cell cycle regulation of Pin1-binding proteins. HeLa cells were harvested from different phases of the cell cycle as described in Fig. 1 and subjected to flow cytometric analysis (bottom) and Far Western analysis using GST–Pin1 as a probe. Equal loading of proteins was shown in each lane by protein staining and anti-Cdc2 immunoblotting analysis (data not shown). Arrows points to Pin1-binding proteins increased at mitosis. (B) Detection of Pin1-binding proteins. GST and GST–Pin1 were incubated with interphase (I) and mitotic (M) HeLa extracts at a final concentration of 20 μm and glutathione beads were added, followed by extensive wash. GST–Pin1 precipitated proteins were separated on SDS-containing gel and stained by Coomassie blue R250. GST did not precipitate any specific Coomassie stainable proteins from either I or M extracts (data not shown). (C) GST–Pin1 can deplete MPM-2 antigens. I and M HeLa cell extracts were either not depleted (Control), or incubated with GST or GST–Pin1 beads. After removing the beads, the supernatants were subjected to immunoblotting analysis using MPM-2. To determine the concentration of Pin1 required to deplete MPM-2 antigens, mitotic extracts were incubated with increasing concentrations of GST–Pin1 and then GST–agarose beads were added to precipitate GST–Pin1-binding proteins. Proteins present in the depleted supernatants, together with control total interphase and mitotic extracts, were subjected to immunoblotting analysis using MPM-2. (D) Coimmunoprecipitation of Pin1 and MPM-2 antigens. Using anti-Pin1 antibodies, Pin1 was immunoprecipitated from I and M lysates in the presence or absence of various phosphatase inhibitors, followed by immunoblotting with MPM-2. Arrows point to the MPM-2 antigens that are immunoprecipitated by Pin1 antibodies. (E) Precipitation of Xenopus MPM-2 antigens by human Pin1. Xenopus interphase (I) extracts were driven into mitosis (M) by addition of nondegradable cyclin B. Both I and M extracts were incubated with agarose beads containing GST or GST–Pin1, followed by immunoblotting analysis with MPM-2 antibody. Arrows point to proteins that are specifically precipitated by GST–Pin1.

Figure 3

Generation of Pin1 mutants unable to bind phosphoproteins. (A) Model for a phosphorylated Ser–Pro dipeptide bound to the active site of Pin1 in the Syn 90 conformation. A basic cluster consisting of conserved Lys-63, Arg-68, and Arg-69 sequesters a sulfate ion in close proximity to the β–methyl group of the Ala residue in the bound Ala–Pro dipeptide. The Phospho–Ser has been modeled on the original Ala in an extended low energy conformation and in the maximal overlap of the extended Phospho–Ser side chain with the sulfate ion. Steric clashes of the Phospho–Ser side chain with the Pin1 active site in the cis or trans conformations would necessitate an active site rearrangement, or a transition of the Phospho–Ser side chain to a higher energy conformation. Atoms in the active site have been color coded for clarity. Oxygen is red, nitrogen is blue, carbon is black, and sulfur is yellow. Syn 90 indicates the conformation of the Phospho–Ser–Pro peptide bond being between cis (0) and trans (180). [Reproduced, with permission, from Ranganathan et al. (1997)] (B) PPIase activity of mutant proteins. Two Pin1 mutants were generated that contain either single Ala substitution at His-59 (Pin1^H59A) or double Ala substitutions at Arg-68 and Arg-69 (Pin1^R68,69A), which are the amino acids implicated in binding the Pro residue or putative phosphate in the substrate, respectively, as shown in A. Both Pin1 and mutants were expressed and purified as GST fusion proteins. Purified Pin1 and the mutants were subjected to PPIase assay using two different substrates; one substrate had an Ala as the amino acid amino-terminal to the Pro (AAPF, open bars), and the second substrate has a Glu amino-terminal to the Pro (AEPF, solid bars). (C) Phosphoprotein-binding activity of mutant proteins. After incubated with I or M extracts, proteins associated with GST–Pin1, GST–Pin1^R68,69A or GST–Pin1^H59A beads were probed with MPM-2. M1 and M2 represent mitotic extracts prepared from two independent experiments. Although the exact intensity of MPM-2 staining in each precipitated protein varies as a result of changes in the phosphorylation state, overall patterns are quite similar in all different experiments.

Figure 4

Pin1 can interact with important mitotic regulators in mitosis-specific and phosphorylation-dependent manner. (A) Binding of Pin1 to human Cdc25C, Plk1, and Myt1. Similar amounts of proteins from I and M HeLa cells were either immunoprecipitated (IP) and immunoblotted using the same anti-Cdc25C or anti-Plk1 antibodies, or subjected to GST bead pulldown assay (Pin1-PP, GST, GST–Pin1), followed by immunoblotting analysis using anti-Cdc25C, anti-Plk1, or anti-Myt1 antibodies. (B) Failure of the Pin1 mutants to bind Cdc25C and Cdc27. GST–glutathione beads containing wild-type and mutant Pin1 proteins were incubated with mitotic extracts and proteins associated with the beads were subjected to immunoblotting analysis using anti-Cdc25C and anti-Cdc27 antibodies. (C) Interaction between Pin1 and Xenopus Cdc27. Xenopus interphase (I) extracts were driven into mitosis (M) and half of the reaction was treated subsequently with calf intestine phosphatase (M + CIP). These three different extracts were incubated with agarose beads containing GST or GST–Pin1, followed by immunoblotting analysis with anti-Cdc27 antibodies. (D) Interaction between Pin1 and other selected mitotic phosphoproteins. The indicated proteins were synthesized by in vitro transcription and translation in the presence of [³⁵S]-methionine and incubated with Xenopus I, M, or M + CIP. These proteins were separated on SDS-containing gels either directly (input) or first precipitated by GST–Pin1 beads (GST–Pin1).

Figure 5

Microinjection of Pin1 protein, but not its mutants, inhibits mitotic division in Xenopus embryos. (A) GST fusion proteins containing wild-type Pin1 or its two mutants were microinjected to into one blastomere of Xenopus embryos at the two–cell stage. The injected embryos were allowed to develop at 18°C for 3 hr (about five cycles) followed by photography of typical embryos. Embryos with a cell cycle arrest had fewer and larger blastomeres on one side. The arrows point to the injected blastomere. (B) GST–Pin1 and the mutants were titrated into the assay described in A except that the embryos were injected at the four–cell stage. The percentage of embryos with a cell cycle block phenotype at each Pin1 concentration was determined and presented graphically.

Figure 6

Pin1, but not the mutants, blocks mitotic entry in Xenopus extracts. (A) Xenopus cytostatic factor (CSF)-arrested extracts containing demembranated sperm and rhodamine–tubulin were activated with 0.4 mm Ca²⁺ at time zero, and 15 min later, Pin1 or the mutants was added to 40 μm and at 110 min the nuclear morphology was examined microscopically. (B) The activity of Cdc2 was followed using histone H1 as a substrate in the CSF extract experiment described in A; 40 μm GST–Pin1 (black squares), 40 μm GST–Pin1^R68,69A (dark blue triangles), and 40 μm GST–Pin1^H59A (brown crossed circles). Also shown are reactions in the same extracts that contained 10 μm GST–Pin1 (green circles) and 40 μm BSA (light blue crossed squares).

Figure 7

Cell cycle-regulated in vivo association of Pin1 and Cdc25 and the effect of mutating Cdc25 phosphorylation sites on the interaction. (A) Cell cycle-dependent interaction between Pin1 and Cdc25. Xenopus eggs were fertilized and assayed for both H1 kinase activity and the Pin1 Cdc25 complex at the indicated times after fertilization. After crushing into a buffer containing 1 μm okadeic acid, aliquots of soluble extracts were used to assay histone H1 kinase activity to monitor cell cycle progression (○), and the remaining extracts were immunoprecipitated with anti-Cdc25 antibodies. The resulting immunoprecipitates were subjected to immunoblotting with anti-Pin1 antibodies (▴). (Left) Pin1 coimmunoprecipitated by anti-Cdc25 antibodies at the two time points (30 and 60 min); (right) relative H1 kinase activity and relative amount of Pin1 that was immunoprecipitated by anti-Cdc25 during the first embryonic cell cycle, with either the maximal amount of Pin1 precipitated or H1 kinase activity being defined as 100%. (B) Failure of Cdc25 mutants to bind Pin1. Wild–type Xenopus Cdc25 (WT), T3 mutant Cdc25 (T48A, T67A, and T138A), and T3S2 mutant Cdc25 (T48A, T67A, T138A, S205A, and S285A) were synthesized by in vitro transcription and translation in the presence of [³⁵S]-methionine and incubated with Xenopus I and M extracts. These proteins were separated on SDS-containing gels either directly (input) or first precipitated by GST–Pin1 beads (GST–Pin1).

Figure 8

Pin1, but not the mutant, directly inhibits the ability of Cdc25 to activate cyclin B/Cdc2. (A) GST–Cdc25 (1 μm), which had been incubated in Xenopus mitotic extracts and purified on glutathione–agarose, was incubated with 0.62 μm of BSA, GST–Pin1, or GST–Pin1^R68,69A. The resulting reaction was incubated for 10 min with beads containing GST–cyclin B/Cdc2 phosphorylated on the inhibitory residues of Cdc2 (Thr-14, Tyr-15). The beads were washed and the activity of cyclin B/Cdc2 was assayed using histone H1 as a substrate. (B) Titration of GST–Pin1 and GST–Pin1^R68,69A into the Cdc25 assay, described in A and in Materials and Methods. (C) Effect of Pin1 on Cdc2 kinase activity. Different concentrations of Pin1 and its mutant protein were added to active cyclin B/Cdc2 complex, followed by assaying H1 kinase activity using the same conditions as described in A and B.