(A) Identification of Ser71 phosphorylation in Pin1 immunoprecipitated from normal breast cells.
(B) Pin1 Ser71 phosphorylation inhibits its PPIase activity. Pin1 and its mutants were phosphorylated by PKA catalytic subunit and then determined their PPIase activities.
(C) Pin1 pSer71 dephosphorylation fully restores its PPIase activity. Dephosphorylation of phosphorylated Pin1 was performed with the Ser/Thr phosphatases PP1, PP2A, PP2B or PP2C.
(D) Pin1-S71D, but not Pin1-S71A mutation abolishes the pSer-Pro specific PPIase activity, without affecting basal Ala-Pro PPIase activity. Results shown are mean ± SEM, n = 3. **, p <0.002.
(E) Structure model of Ser71 inhibitory phosphorylation, with a full view in Figure S1B.
(F) Pin1-S71D and Pin1-S71E mutations abolish the ability to activate cyclin D1 promoter in cells. HeLa cells were co-transfected with Pin1, its mutants, reporter constructs or vector control, followed by assaying the luciferase activity. Results shown are mean ± SEM, n = 3. **, p <0.001.
(G and H) Pin1-S71D mutation abolishes the ability to stabilize endogenous cyclin D1 protein in cells. Pin1−/− MEFs expressing Pin1 or its S71 mutants were treated with cycloheximide, followed by immunoblot with anti-cyclin D1, Pin1 or β-actin antibodies (G). Cyclin D1 levels were semi- quantitated using β-actin as a loading control and the relative levels at time 0 defined as 1 (H).

(A and B) Co-immunoprecipitation of expressed DAPK1 and Pin1 in vivo. 293T cells expressing FLAG-DAPK1 or K42A mutant and HA-Pin1 were subjected to immunoprecipitation with anti-HA (A) or FLAG (B) antibodies, followed by immunoblot with anti-FLAG or HA antibodies.
(C) DAPK1 and Pin1 interaction in vitro. 293T cells expressing DAPK1 or its mutants were subjected to GST pulldown assay.
(D) Cell cycle-independent DAPK1 and Pin1 interaction. 293T cells expressing DAPK1 were incubated with or without nocodazole before GST pulldown assay.
(E) DAPK1 binding to the PPIase domain, but not WW domain of Pin1. 293T cells expressing DAPK1 were subjected to GST pulldown assay.
(F) Pin1 binding to the Cyto domain of DAPK1. 293T cells expressing DAPK1 or its truncated mutants were subjected to GST pulldown assay.
(G) A deletion of the Cyto domain of DAPK1 fails to bind to Pin1. 293T cells expressing DAPK1 or its Cyto deletion mutants were subjected to GST pulldown assay.
(H) The Cyto domain of DAPK1 is sufficient to interact with Pin1. 293T cells expressing DAPK1-Cyto were subjected to GST pulldown assay.
(I) Schematic representation of full length DAPK1 and its truncated mutants.

(A) DAPK1 phosphorylates Pin1 in vitro. Purified recombinant DAPK1 was incubated with GST Pin1 or MLC and ³²P-ATP, detected by autoradiography.
(B) Failure of kinase-impaired DAPK1 mutant to phosphorylate Pin1. HeLa cells expressing DAPK1, DAPK1^K42A or vector control were immunoprecipitated with anti-FLAG antibodies and then incubated with His-Pin1 and ³²P-ATP, detected by autoradiography.
(C) Time-dependent phosphorylation of Pin1 by DAPK1.
(D) DAPK1 phosphorylates Pin1 PPIase domain, but not WW domain. Purified recombinant DAPK1 was incubated with GST-Pin1, WW or PPIase domain and ³²P-ATP, detected by autoradiography.
(E) DAPK1 phosphorylates Pin1 on Ser71, but not Ser16 in vitro.
(F) DAPK1 does not phosphorylate Pin1 on Ser72 in vitro.
(G) DAPK1 phosphorylates Pin1 on Ser71 in vitro. Purified recombinant DAPK1 was incubated with GST-Pin1, followed by immunoblotting with anti-pSer71, C-terminus, Ser16 or pSer16 Pin1 antibodies.
(H) DAPK1 phosphorylates Pin1 on Ser71 in vivo. HeLa cell expressing FLAG-DAPK1 or K42A mutant and HA-Pin1 were subjected to immunoprecipitation with anti-HA, followed by immunoblotting with anti-pSer71 Pin1 antibodies.
(I) Failure of DAPK1 phosphorylation to phosphorylate Pin1 S71A mutant. HeLa cells expressing FLAG-DAPK1^ΔCaM and HA-Pin1 or its mutant S71A were induced FLAG-DAPK1^ΔCaM expression using doxycycline and then subjected to immunoprecipitation with anti-HA antibodies, followed by immunoblot with anti-pSer71 Pin1 antibodies.
(J) Endogenous phosphorylation of Pin1 on Ser71 by DAPK1. Various breast cell lines were subjected to immunoprecipitation with anti-Pin1, followed by immunoblot with anti-pSer71 Pin1 or DAPK1 antibodies. * Immunoglobulin light chain.
(K) DAPK1 knockdown abolishes endogenous Pin1 on Ser71 phosphorylation. MCF10A cells stably expressing DAPK1 shRNAs were subjected to immunoprecipitation with pre-immune serum or anti-Pin1, followed by immunoblot with anti-pSer71 Pin1 or DAPK1 antibodies.
(L) PKA does not phosphorylate Pin1 on Ser71 in vivo. DAPK1 shRNA MCF10A cells were treated with H89, forskolin or both, followed by immunoprecipitation with anti-Pin1 and immunoblot with anti-pSer71 Pin1 antibodies.

(A and B) DAPK1 inhibits the ability of Pin1 to stabilize cyclin D1 protein. Pin1−/− MEFs expressing Pin1 and DAPK1^ΔCaM, DAPK1^K42A or vector control were treated with cycloheximide, followed by immunoblot analysis (A) and semi-quantitated using β-actin (B). Results shown are mean ± SEM, n = 3.
(C) DAPK1 inhibits the ability of Pin1 to activate cyclin D1 promoter in cells. HeLa cells were transiently transfected with Pin1, reporter constructs and DAPK1, its mutants or vector control, followed by assaying the luciferase activity. Results shown are mean ± SEM, n = 3. **, p <0.01.
(D) DAPK1 and Pin1 binding is required for DAPK1 to inhibit Pin1-mediated cyclin D1 activation. Results shown are mean ± SEM, n = 3. **, p <0.01.
(E) DAPK1 prevents Pin1 nuclear localization. Cells expressing DAPK1, its mutants or control were immunostained with anti-Pin1 (green), anti-DAPK1 (red) antibodies and DAPI (blue). White and yellow arrows point to DAPK1^ΔCaM-transfected and non-transfected cells, respectively.
(F) Ser71 phosphorylation regulates Pin1 localization. Cells inducibly expressing Pin1 or its Ser71 mutants or inducibly expressing DAPK1 with Pin1^S71A overexpression were stained with anti-HA (green) or FLAG (red) antibodies and DAPI (blue).

(A and B) S71D, but not S71A abolishes the ability of Pin1 to induce centrosome amplification. NIH3T3 cells stably expressing Pin1, its Ser71 mutants or control were arrested at the G1/S boundary by aphidicolin. Cells were stained with anti-γ-tubulin antibody (red) and DAPI (blue). Bar, 10 μm (A). Cells containing >2 centrosomes were scored in 300 transfected cells (B). Results shown are mean ± SEM, n = 3. **, p <0.001.
(C and D) DAPK1 inhibits Pin1-induced centrosome amplification. Stable cell lines were arrested at the G1/S boundary by aphidicolin in the presence or absence of doxycycline. Cells were stained with anti-γ-tubulin antibody (red) and DAPI (blue). Bar, 10 μm (C). Cells containing >2 centrosomes were scored in 300 transfected cells (D). Results shown are mean ± SEM, n = 3.

(A and B) Ser71 phosphorylation suppresses the ability of Pin1 to induce the formation of multipolar spindles. NIH3T3 cells stably expressing Pin1 or its Ser71 mutants were released from the aphidicolin block for 24 hr, then fixed and co-stained with anti-α-tubulin (green) and anti-γ-tubulin (red) antibodies followed by DAPI staining (blue). Representative mitotic cells with bipolar or multipolar spindle poles were shown. Bar, 10 μm (A). Cells containing >2 mitotic spindle poles were scored in 200 mitotic cells (B). Results shown are mean ± SEM, n = 3. **, p <0.01.
(C–F) Ser71 phosphorylation abrogates the ability of Pin1 to induce anchorage-dependent cell growth on plastic plates (C and D) and soft agar (E and F). Stable NIH3T3 cells were released from the aphidicolin block and seeded on plastic plates (C) or in soft agar (E) for 3 weeks, followed by crystal violet staining (C) or P-iodonitrotetrazolium violet staining (E). The number of colonies formed per 1000 cells was scored (D, F). Colony numbers are mean ± SEM, n = 3.**, p <0.002.
(G and H) Effect of DAPK1 and Pin1 on wound-healing migration. MDA-MB-231 cells expressing Pin1 shRNA, DAPK1 shRNA or both were assayed for wound-healing migration, and cell migration into wounds was monitored by time-lapse microscopy. Still images were captured at the indicated times after wounding. Bar, 200 μm. Results shown are mean ± SEM, n = 3. **, p <0.01.

(A and B) Serial sections of tissue arrays of 37 normal breast specimen were subjected to immunohistochemistry using anti-DAPK1 (upper panel) or anti-pSer71 Pin1 antibodies (low panel), and visualized by the DAB staining (A). In each sample, DAPK1 expression and pSer71 levels were semi-quantified in a double-blind manner as high or low according to the standards presented in (A) and summarized in (B). Their correlation was analyzed by Spearman rank correlation test (P<0.001).
(C–E) Serial sections of tissue arrays of 78 breast cancer specimen were subjected to immunohistochemistry using anti-DAPK1 (upper panel), anti-pSer71 Pin1 antibodies (middle panel) and visualized by the DAB staining (brown) and different sections of the same tissues immunostained with anti-γ-tubulin antibodies (low panel) and visualized by Alexa488-conjugated secondary antibody (red) along with DAPI (blue) (C). In each sample, DAPK1 expression and pSer71 levels were semi-quantified in a double-blind manner as high or low according to the standards presented in (C) and summarized in (D). The ratio of centrosomes to nuclei was scored under a fluorescent microscope (C) and summarized in (E). Their correlation was analyzed by Spearman rank correlation test (P<0.001).