Treatment of these human leukemic cell lines in culture with two different Pim inhibitors AZD1208 and LGB321 markedly reduced IRS1S1101 phosphorylation (Figure ?(Figure6A).6A). pan-Pim inhibitor administered Istaroxime to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy. K/RXRHXpS/pT could help in identifying potential substrates of Istaroxime Pim protein kinase. This analysis led to the discovery that IRS1 contains a highly conserved Istaroxime Pim phosphorylation sequence at S1101. Given the role of Pim in regulating a signal transduction pathway related to metabolism [5, 14, 15], this potential substrate was investigated further as a potential biomarker of Pim kinase activity. RESULTS Pim protein kinases regulate IRS1 phosphorylation To search for proteins possessing similar phosphorylation consensus sites, we utilized the NetworKIN resource, a comprehensive database of predicted kinaseCsubstrate relations derived from the human NAK-1 phosphoproteome and integrating interaction networks from the Phospho.ELM, PhosphoSite and STRING databases [14, 16, 17]. The NetworKIN database [18] was queried using AKT and Pim2 kinases Istaroxime for potential substrates. This uncovered 1,247 predicted substrates for Pim2 and 598 for AKT. Among them, 28 proteins contained RXRHXpS/pT Pim phosphorylation recognition motif. This highly conserved consensus sequences was observed on human IRS1 S1101 (S1097 in mouse) and IRS2 S1149 (S1138 in mouse). The context and ranking scores for these target positions were amongst the highest (IRS1 0.983/13.634 and IRS2 0.982/13.62), indicating that IRS1/2 were potential substrates for Pim kinases. To investigate whether IRS is an substrate for Pim protein kinases, MEF cells derived from wild type (WT) and triple knockout of Pim1, Pim2 and Pim3 (TKO) FVB mice were examined. Western blot analysis demonstrated that phosphorylated IRS1 protein expression was undetectable in TKO cells when protein was probed with anti-phospho S1101 IRS1 antibody (Figure ?(Figure1A;1A; lane 1 and 2). Western blot analysis of kidney tissues from WT and TKO mice also demonstrated that IRS1 phosphorylation was markedly reduced in TKO mouse tissues (Figure ?(Figure1B).1B). To identify whether one or all of the three PIM isoforms were regulating the phosphorylation of IRS1, each isoform was transduced into TKO cells using lentiviruses producing Pim1, Pim2 or Pim3. Each of the three isoforms was sufficient to induce the phosphorylation of IRS1 on S1101 (Figure ?(Figure1A;1A; lane 3 to 6). Consistent with these results, the depletion of each Pim kinase isoform individually using siRNA in the prostate cancer cell line PC3-LN4 cells did not decrease IRS1 phosphorylation, but the knockdown of all three isoforms abolished the phosphorylation of the IRS1 protein (Figure ?(Figure1C).1C). Similarly, depletion of Pim1, 2 and 3 by siRNAs in non-small cell lung carcinoma cell line (A549) and a cervical cancer cell line (HeLa) abolished phosphorylation of IRS proteins on S1101. Open in a separate window Figure 1 Expression of Pim1, 2, and 3 kinases control IRS1S1101 phosphorylation(A) IRS1S1101 (IRS1S1097 in mouse) expression levels in WT, TKO, and TKO MEF cells expressing a single isoform of Pim kinase. (B) IRS1S1101 (IRS1S1097 in mouse) expression levels in kidney tissues of WT and TKO mice (two mice for each). Cell lysates of IRS1 expressing HEK293T transfectants were used as positive controls. (C) PC3-LN4, A549 and HeLa cells were transfected with siRNA targeting Pim1, 2, 3 or all three Pims and analyzed after 48 hr. (D) Prostate cancer PC-3 cells expressing tet-inducible Pim1, and human prostate fibroblast cell lines BHPrS1 and WPMY1 expressing tet-inducible Pim1 were stimulated with doxycycline Istaroxime at the indicated doses for 48 hr. Western blots were probed with the listed antibodies. (E) HA-tagged wild type (wt) and.