Fig 1: Expression of PIM1 upregulated genes are dependent on 14-3-3 ζ and AR expression and activity.a Knockdown of 14-3-3 ζ results in decrease in expression of PIM1-induced genes involved in cell migration and invasion (DYNC1I1: 0.68 + /− 0.23, p = 0.030. MMP7:0 .072 + /− 0.038, p < 0.0001. MMP10: 0.70 + /− 0.25, p = 0.025. COL4A6: 0.46 + /− 0.23, p = 0.0034. TLL2: 0.83 + /− 0.21, p = 0.14. n = 4 biological replicates, two sided T test). b Knockdown of AR results in reduction in expression of some of these genes, but an increase in expression of others, indicating that AR may act as an activator at some genes and a repressor at others (DYNC1I1: 1.25 + /− 0.37, p = 0.30. MMP7: 0.24 + /− 0.16, p = 0.0011. MMP10: 3.44 + /− 0.71, p = 0.0040. COL4A6: 0.24 + /− 0.091, p = 0.00013. TLL2: 13.76 + /− 8.72, p = 0.064. n = 3 biological replicates, two sided T test). c Cells in charcoal-stripped media treated with the synthetic androgen R1881 show complementary gene expression changes to cells with AR knockdown (DYNC1I1: 1.43 + /− 0.16, p = .011. MMP7: 1.22 + /− 0.16, p = 0.075. MMP10: 0.40 + /− 0.089, p = 0.00031. COL4A6: 1.78 + /− 0.14, p = 0.00066. TLL2: 0.69 + /− 0.14, p = 0.016. n = 3 biological replicates, two sided T test). c 25 uM enzalutamide treatment for 24 h for the most part replicates AR siRNA knockdown (DYNC1I1: 0.81 + /− 0.36, p = 0.41. MMP7: 0.69 + /− 0.13, p = 0.013. MMP10: 2.18 + /− 0.82, p = 0.067. COL4A6: 0.35 + /− 0.17, p = 0.0027. TLL2: 1.19 + /− 0.22, p = 0.21. n = 3 biological replicates, two sided T test). Error bars refer to standard deviation.
Fig 2: AR and 14-3-3 ζ co-occupy chromatin at MMP7 and TLL2 in a PIM1-dependent manner.a ChIP-seq traces for 14-3-3 ζ and pS213 AR at MMP7 and TLL2 in control and PIM1 over-expressing LNCaP cells. b Validation of 14-3-3 ζ and AR binding at MMP7 and TLL2 by ChIP-PCR. 14-3-3 ζ shows increased binding in PIM1 over-expressing cells (MMP7: 33.47 + /− 2.64 vs. 21.07 + /− 1.22, p = 0.0029, TLL2: 63.95 + /− 13.81 vs. 32.62 + /− 1.43, p = .0248, 3 technical replicates, two sided T test). c ChIP-PCR of 14-3-3 ζ binding at MMP7 and TLL2 in PIM1 over-expressing cells after treatment with the PIM1 inhibitor SGI1776 (7.5 uM for 24 h) and enzalutamide (25 uM for 24 h). 14-3-3 ζ binding is reduced in the presence of both treatments for both genes (for MMP7: Veh: 34.95 + /− 1.27 vs. SGI1776 24.09 + /− 4.76, p = 0.01167, n = 3, two sided T test, Veh: 34.95 + /− 1.27 vs. Enz: 13.02 + /− 5.18, p = 0.0011, n = 3, two sided T test. For TLL2: Veh: 50.88 + /− 5.78 vs. SGI1776 15.78 + /− 1.02, p = 0.00057, n = 3, two sided T test, and Veh: 50.88 + /− 5.78 vs. Enz: 8.06 + /− 1.19, p = 0.00025, n = 3, two sided T test.). d ChIP-PCR of AR binding at MMP7 and TLL2 in PIM1 over-expressing cells after treatment with the PIM1 inhibitor SGI1776 (7.5 uM for 24 h) and enzalutamide (25 uM for 24 h). AR binding is reduced in the presence of both treatments (for MMP7: Veh: 181.98 + /− 0.53 vs. SGI1776: 103.49 + /− 6.46, p = 0.000332, n = 3, two sided T test, and Veh: 181.98 + /− 0.53 vs. Enz: 6.78 + /− 0.70, p = 0.000005, n = 3, two sided T test. For TLL2: Veh: 130.72 + /− 11.96 vs. SGI1776 54.62 + /− 6.20, p = 0.00012, n = 3, two sided T test, and Veh: 130.72 + /− 11.96 vs. Enz: 9.64 + /− .57, p < .0001, n = 3, two sided T test). Error bars refer to standard deviation.
Fig 3: Model for PIM1-mediated AR co-regulation by 14-3-3 ζ.PIM1 phosphorylation of the AR and 14-3-3 ζ enhances their interaction and shifts their occupancy on chromatin, resulting in 14-3-3 ζ co-regulation of AR, likely by recruiting other AR co-regulators such as hnRNPK and TRIM28. Nucleus image used from smart.servier.com.
Fig 4: PIM1 phosphorylates the AR and 14-3-3 ζ and coordinates their interaction.a Inducible PIM1 over-expression in LNCaP cells results in an increase in phosphorylation of AR at S213 (including relative abundance of pS213 AR quantified). b 14-3-3 ζ functions as an AR co-activator in LNCaP cells. 14-3-3 ζ knockdown in ARR3-luciferase assay leads to reduction in AR transcriptional activity (33.10 + /− 3.008 vs. 14.39 + /− 0.396, p = .000017, n = 4 biological replicates, two sided t test). c PIM1 phosphorylates 14-3-3 ζ in vitro, predominately at S64. d The PIM1 phosphorylation site on AR, S213, lines up with the 14-3-3 consensus interaction site. e 14-3-3 ζ co-immunoprecipitates with AR, particularly in the presence of PIM1 over-expression, and less so with AR phosphorylation mutant S213A (including relative abundance of AR co-IP quantified). f AR co-immunoprecipitates with WT 14-3-3 ζ in LNCaP cells, and less so with 14-3-3 ζ phosphorylation mutant S64A (including relative abundance of 14-3-3 ζ co-IP quantified). Error bars refer to standard deviation.
Fig 5: AR and 14-3-3 ζ interacting proteins hnRNPK and TRIM28 are involved in PIM1-dependent gene expression.a Knockdown of hnRNPK and TRIM28 result in alterations of gene expression for PIM1-dependent gene panel (for hnRNPK knockdown: DYNC1I1: 2.24 + /− 0.80, p = .055, MMP7: 0.76 + /− 0.088, p = 0.0087, MMP10: 1.67 + /− 0.62, p = .13, COL4A6: 0.37 + /− 0.16, p = .0023, TLL2: 0.58 + /− 0.032, p < .0001. For TRIM28 knockdown: DYNC1I1: 1.50 + /− 0.43, p = .12, MMP7: 2.01 + /− 0.20, p = 0.00086, MMP10: 1.69 + /− 0.11, p = .00049, COL4A6: 0.96 + /− 0.49, p = 0.90, TLL2: 1.05 + /− .087, p = 36). b ChIP of hnRNPK and TRIM28 at MMP7 reveals modest occupancy of hnRNPK at MMP7 AR occupancy site, and substantial TRIM28 occupancy. c Co-immunoprecipitation of AR with TRIM28 reveals a reduction in AR interaction with TRIM28 in PIM1 over-expressing cells. Error bars refer to standard deviation.
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