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. Author manuscript; available in PMC: 2012 Apr 22.
Published in final edited form as: Mol Cell. 2011 Apr 14;42(2):147–159. doi: 10.1016/j.molcel.2011.03.005

Figure 3. DAPK1 phosphorylates Pin1 on Ser71 in vitro and in vivo.

Figure 3

(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, DAPK1K42A 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-DAPK1ΔCaM and HA-Pin1 or its mutant S71A were induced FLAG-DAPK1ΔCaM 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.

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