Pim-1 kinase expression during murine mammary development

Pim-1 kinase phosphorylates substrates whose activities are linked to proliferation, survival, differentiation, and apoptosis. Although pim-1 is induced by hormones and cytokines, the hormonal control and contribution of Pim-1 to mammary gland development have not been evaluated. We examined Pim-1 expression in mammary cell lines, investigated whether Pim-1 levels could be altered in breast epithelia by mammogenic hormones, and evaluated Pim-1 expression during mammary development. We found that Pim-1 was elevated in most mammary carcinoma cell lines and progesterone increased Pim-1 protein to some extent in non-tumorigenic mammary epithelia. Pim-1 expression in situ was consistent with the documented profile of progesterone activity in mouse mammary glands. Pim-1 nuclear localization correlated with cytoplasmic distribution for its substrate, p21(CIP/Waf1), and we found that Pim-1 and p21 associate in vitro. Our results suggest that Pim-1 expression may be regulated by progesterone during mammary development and Pim-1 associates with p21 in mammary epithelial cells.     

Pim-2 phosphorylation of p21Cip1/WAF1 enhances its stability and inhibits cell proliferation in HCT116 cells

Pim-2 kinase is one of the three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21^Cip1/WAF1 (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21 and results in enhanced levels of both exogenous and endogenous p21 proteins. Knockdown of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.     

EBNA3C Augments Pim-1 Mediated Phosphorylation and Degradation of p21 to Promote B-Cell Proliferation

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, can latently infect the human population. EBV is associated with several types of malignancies originating from lymphoid and epithelial cell types. EBV latent antigen 3C (EBNA3C) is essential for EBV-induced immortalization of B-cells. The Moloney murine leukemia provirus integration site (PIM-1), which encodes an oncogenic serine/threonine kinase, is linked to several cellular functions involving cell survival, proliferation, differentiation, and apoptosis. Notably, enhanced expression of Pim-1 kinase is associated with numerous hematological and non-hematological malignancies. A higher expression level of Pim-1 kinase is associated with EBV infection, suggesting a crucial role for Pim-1 in EBV-induced tumorigenesis. We now demonstrate a molecular mechanism which reveals a direct role for EBNA3C in enhancing Pim-1 expression in EBV-infected primary B-cells. We also showed that EBNA3C is physically associated with Pim-1 through its amino-terminal domain, and also forms a molecular complex in B-cells. EBNA3C can stabilize Pim-1 through abrogation of the proteasome/Ubiquitin pathway. Our results demonstrate that EBNA3C enhances Pim-1 mediated phosphorylation of p21 at the Thr¹⁴⁵ residue. EBNA3C also facilitated the nuclear localization of Pim-1, and promoted EBV transformed cell proliferation by altering Pim-1 mediated regulation of the activity of the cell-cycle inhibitor p21/WAF1. Our study demonstrated that EBNA3C significantly induces Pim-1 mediated proteosomal degradation of p21. A significant reduction in cell proliferation of EBV-transformed LCLs was observed upon stable knockdown of Pim-1. This study describes a critical role for the oncoprotein Pim-1 in EBV-mediated oncogenesis, as well as provides novel insights into oncogenic kinase-targeted therapeutic intervention of EBV-associated cancers.     

Pim-1 negatively regulates the activity of PTP-U2S phosphatase and influences terminal differentiation and apoptosis of monoblastoid leukemia cells

The levels of Pim-1, a serine/threonine kinase, increase during phorbol myristate acetate (PMA)-induced myeloid cell differentiation. The tyrosine phosphatase PTP-U2S is also associated with PMA-induced differentiation of myeloid cells and has been shown to enhance differentiation and the onset of apoptosis. PTP-U2S contains a Pim-1 phosphorylation consensus sequence, KKRKLTN, which is efficiently phosphorylated by Pim-1. Immunoprecipitated PTP-U2S from U937 cells was phosphorylated by recombinant Pim-1, resulting in a decrease in its phosphatase activity. During PMA-induced differentiation, U937 cells transfected with the dominant negative Pim-1 underwent rapid differentiation and accelerated apoptosis. The opposite effect was observed for wild-type Pim-1. Our results, therefore, provide compelling evidence that Pim-1 functions to negatively regulate PMA-induced differentiation in part through the phosphorylation of PTP-U2S. Together these data suggest that Pim-1 phosphorylates PTP-U2S in vivo to decrease the phosphatase activity that may be necessary to prevent the premature onset of apoptosis following differentiation.     

Pim-1 kinase-dependent phosphorylation of p21Cip1/WAF1 regulates its stability and cellular localization in H1299 cells

Previous studies from our laboratory showed that p21Cip1/WAF1 can be phosphorylated by Pim-1 kinase in vitro, implying that part of the function of Pim-1 might involve influencing the cell cycle. In the present study, site-directed mutagenesis and phosphorylated-specific antibodies were used as tools to identify the sites phosphorylated by Pim-1 and the consequences of this phosphorylation. What we found was that Pim-1 can efficiently phosphorylate p21 on Thr145 in vitro using recombinant protein and in vivo in intact cells. Unexpectedly, we found that Ser146 is a second site that is phosphorylated in vivo, but this phosphorylation event seems to be an indirect result of Pim-1 expression. More importantly, the consequences of phosphorylation of either Thr145 or Ser146 are distinct. When p21 is phosphorylated on Thr145, it localizes to the nucleus and results in the disruption of the association between proliferating cell nuclear antigen and p21. Furthermore, phosphorylation of Thr145 promotes stabilization of p21. On the other hand, when p21 is phosphorylated on Ser146, it localizes primarily in the cytoplasm and the effect of phosphorylation on stability is minimal. Cotransfection of wild-type Pim-1 with p21 increases the rate of proliferation compared with cotransfection of p21 with kinase-dead Pim-1. Knocking down Pim-1 expression greatly decreases the rate of proliferation of H1299 cells and their ability to grow in soft agar. These data suggest that Pim-1 overexpression may contribute to tumorigenesis in part by influencing the cellular localization and stability of p21 and by promoting cell proliferation.     

Mitogen-activated protein kinase kinase 1 (MEK1) stabilizes MyoD through direct phosphorylation at tyrosine 156 during myogenic differentiation

Previously, we reported that mitogen-activated protein kinase kinase 1 (MEK1) activated in the mid-stage of skeletal muscle differentiation promotes myogenic differentiation. To elucidate the molecular mechanism, we investigated an activity of MEK1 for MyoD. Activated MEK1 associates with MyoD in the nucleus of differentiating myoblasts. In vitro kinase assay using active MEK1, a (32)P-labeled protein band corresponding to GST-MyoD was observed but not to mutant GST-MyoD-Y156F. Tyrosine phosphorylation of endogenous MyoD was detected with a specific anti-pMyoD-Y156 antibody; however, this response was blocked by PD184352, a MEK-specific inhibitor. These results indicate that activated MEK1 phosphorylates the MyoD-Y156 residue directly. Interestingly, the protein level of mutant MyoD-Y156F decreased compared with that of wild type but was recovered in the presence of lactacystin, a proteasome inhibitor. The protein level of MyoD-Y156E, which mimics phosphorylation at Tyr-156, was above that of wild type, indicating that the phosphorylation protects MyoD from the ubiquitin proteasome-mediated degradation. In addition, the low protein level of MyoD-Y156F was recovered over that of wild type by an additional mutation at Leu-164, a critical binding residue of MAFbx/AT-1, a Skp, Cullin, F-box (SCF) E3-ubiquitin ligase. The amount of MyoD co-precipitated with MAFbx/AT-1 also was reduced in the presence of active MEK1. Thus, these results suggested that the phosphorylation probably interrupts the binding of MAFbx/AT-1 to MyoD and thereby increases its stability. Collectively, our results suggest that MEK1 activated in differentiating myoblasts stimulates muscle differentiation by phosphorylating MyoD-Y156, which results in MyoD stabilization.     

Pim-1 associates with protein complexes necessary for mitosis

The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.     

Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells

Amplification or overexpression of HER-2/neu in cancer cells confers resistance to apoptosis and promotes cell growth. The cellular localization of p21Cip1/WAF1 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Here we show that HER-2/neu-mediated cell growth requires the activation of Akt, which associates with p21Cip1/WAF1 and phosphorylates it at threonine 145, resulting in cytoplasmic localization of p21Cip1/WAF1. Furthermore, blocking the Akt pathway with a dominant-negative Akt mutant restores the nuclear localization and cell-growth-inhibiting activity of p21Cip1/WAF1. Our results indicate that HER-2/neu induces cytoplasmic localization of p21Cip1/WAF1 through activation of Akt to promote cell growth, which may have implications for the oncogenic activity of HER-2/neu and Akt.     

Plk1 phosphorylation of TRF1 is essential for its binding to telomeres

In a search for Polo-like kinase 1 (Plk1) interaction proteins, we have identified TRF1 (telomeric repeat binding factor 1) as a potential Plk1 target. In this communication we report further characterization of the interaction. We show that Plk1 associates with TRF1, and Plk1 phosphorylates TRF1 at Ser-435 in vivo. Moreover, Cdk1, serving as a priming kinase, phosphorylates TRF1 to generate a docking site for Plk1 toward TRF1. In the presence of nocodazole, ectopic expression of wild type TRF1 but not TRF1 with alanine mutation in the Plk1 phosphorylation site induces apoptosis in cells containing short telomeres but not in cells containing long telomeres. Unexpectedly, down-regulation of TRF1 by RNA interference affects cell proliferation and results in obvious apoptosis in cells with short telomeres but not in cells with long telomeres. Importantly, we observe that telomeric DNA binding ability of TRF1 is cell cycle-regulated and reaches a peak during mitosis. Upon phosphorylation by Plk1 in vivo and in vitro, the ability of TRF1 to bind telomeric DNA is dramatically increased. These results demonstrate that Plk1 interacts with and phosphorylates TRF1 and suggest that Plk1-mediated phosphorylation is involved in both TRF1 overexpression-induced apoptosis and its telomeric DNA binding ability.     

Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site

Constitutive expression of the Pim-1 kinase prolongs survival of cytokine-deprived FDCP1 cells, partly via maintenance of Bcl-2 expression. Here, we show that Pim-1 colocalizes and physically interacts with the pro-apoptotic Bad protein and phosphorylates it in vitro on serine 112, which is a gatekeeper site for its inactivation. Furthermore, wild-type Pim-1, but not a kinase-deficient mutant, enhances phosphorylation of this site in FDCP1 cells and protects cells from the pro-apoptotic effects of Bad. Our results suggest that phosphorylation of Bad by Pim-1 is one of several mechanisms via which the Pim-1 kinase can enhance Bcl-2 activity and promote cell survival.     

p70 S6K1 nuclear localization depends on its mTOR-mediated phosphorylation at T389, but not on its kinase activity towards S6

The protein kinase p70 S6K1 is regulated in response to cytokines, nutrients and growth factors, and plays an important role in the development of a variety of human diseases. Mammalian target of rapamycin (mTOR) is known to phosphorylate and thereby activate p70 S6K1. p70 S6K1 phosphorylates different cytoplasmic and nuclear substrates involved in the regulation of protein synthesis, cell cycle, cell growth and survival. Recently, we have shown that mTOR-mediated phosphorylation of p70 S6K1 at T389 also regulates its nucleocytoplasmic localization. Since this phosphorylation is associated with its kinase activity the question whether p70 S6K1 phosphorylation or kinase activity is essential for its proper localization remained elusive. Recently, the chemical compound PF-4708671 has been demonstrated to block p70 S6K1 kinase activity while inducing its phosphorylation at T389. This potential of PF-4708671 to separate p70 S6K1 activity from its T389 phosphorylation allowed us to demonstrate that the proper nucleocytoplasmic localization of this kinase depends on its mTOR-mediated phosphorylation but not on its kinase activity. These findings provide important insights into the regulation of p70 S6K1 and allow a more detailed understanding of subcellular enzyme localization processes.     

Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cells

SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S -transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro . Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation.     

14-3-3 Protein mediates phosphorylation of microtubule-associated protein tau by serum- and glucocorticoid-induced protein kinase 1

The microtubule-associated protein, tau, is involved in numerous neuronal processes such as vesicle transport, microtubule-plasma membrane interaction and the intracellular localization of proteins. Tau is known to be phosphorylated by several kinases such as mitogen activated protein kinase, microtubule affinity regulating kinase, and protein kinase A. We found a putative serum- and glucocorticoid-induced protein kinase 1 (SGK1) phosphorylation site within the 207GSRSRTPSLP216 tau amino acid sequence. We report here that SGK1 phosphorylates Ser214 of Tau. Using a pull-down assay, we found that 14-3-3q interacts with SGK1 and tau to form a ternary protein complex that leads to phosphorylation of tau. 14-3-3 and phosphorylated tau were mainly co-localized in the nucleus of COS-1 cells. These results demonstrate that 14-3-3 scaffolds tau with SGK1 to facilitate the phosphorylation of tau at Ser214 and to regulate its subcellular localization.     

Regulation of Skp2 Levels by the Pim-1 Protein Kinase

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF^Skp2 ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser⁶⁴ and Ser⁷², we have identified Thr⁴¹⁷ as a unique Pim-1 phosphorylation target. Phosphorylation of Thr⁴¹⁷ controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.     

The c-Src tyrosine kinase regulates signaling of the human DF3/MUC1 carcinoma-associated antigen with GSK3 beta and beta-catenin

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in most human carcinomas. The cytoplasmic domain of MUC1 interacts with glycogen synthase kinase 3 beta (GSK3 beta) and thereby decreases binding of MUC1 and beta-catenin. The present studies demonstrate that MUC1 associates with the c-Src tyrosine kinase. c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif located between sites involved in interactions with GSK3 beta and beta-catenin. The results demonstrate that the c-Src SH2 domain binds directly to pYEKV and inhibits the interaction between MUC1 and GSK3 beta. Moreover and in contrast to GSK3 beta, in vitro and in vivo studies demonstrate that c-Src-mediated phosphorylation of MUC1 increases binding of MUC1 and beta-catenin. The findings support a novel role for c-Src in regulating interactions of MUC1 with GSK3 beta and beta-catenin.     

Structure and substrate specificity of the Pim-1 kinase

The Pim kinases are a family of three vertebrate protein serine/threonine kinases (Pim-1, -2, and -3) belonging to the CAMK (calmodulin-dependent protein kinase-related) group. Pim kinases are emerging as important mediators of cytokine signaling pathways in hematopoietic cells, and they contribute to the progression of certain leukemias and solid tumors. A number of cytoplasmic and nuclear proteins are phosphorylated by Pim kinases and may act as their effectors in normal physiology and in disease. Recent crystallographic studies of Pim-1 have identified unique structural features but have not provided insight into how the kinase recognizes its target substrates. Here, we have conducted peptide library screens to exhaustively determine the sequence specificity of active site-mediated phosphorylation by Pim-1 and Pim-3. We have identified the major site of Pim-1 autophosphorylation and find surprisingly that it maps to a novel site that diverges from its consensus phosphorylation motif. We have solved the crystal structure of Pim-1 bound to a high affinity peptide substrate in complexes with either the ATP analog AMP-PNP or the bisindolylmaleimide kinase inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide HCl. These structures reveal an unanticipated mode of recognition for basic residues upstream of the phosphorylation site, distinct from that of other kinases with similar substrate specificity. The structures provide a rationale for the unusually high affinity of Pim kinases for peptide substrates and suggest a general mode for substrate binding to members of the CAMK group.     

Akt-Dependent Phosphorylation of p21Cip1 Regulates PCNA Binding and Proliferation of Endothelial Cells

The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21(Cip1) at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21(Cip1) at Thr 145 prevents the complex formation of p21(Cip1) with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21(Cip1) at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21(Cip1) and attenuates the Cdk2 inhibitory activity of p21(Cip1). Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21(Cip1) phosphorylation is not caused by altered intracellular localization of p21(Cip1). As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21(Cip1), whereas the nonphosphorylatable p21(Cip1) T145A construct arrests cells in G(0) phase. These data suggest that the modulation of p21(Cip1) cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.     

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.