PIM-2 is an independent regulator of chondrocyte survival and autophagy in the epiphyseal growth plate

The overall goal of the investigation was to examine the activity and role of the PIM serine/threonine protein kinases in the growth plate. We showed for the first time that PIM-2 was highly expressed in epiphyseal chondrocytes and that the kinase was required for critical activities linked to cell survival. These activities were independent of those mediated by Akt-1. It was noted that PIM-2 protected chondrocytes from rapamycin sensitized (TOR inhibited) cell death. Since inhibition of mTOR caused autophagy, we examined the autophagic response of PIM-2 silenced cells. We showed that PIM-2 promoted expression and organization of autophagic proteins LC3, and Beclin-1 and enhanced lysosomal acidification. At the same time, PIM-2 modulated the activity of a key regulator of apoptosis, BAD. Since BAD inhibition and Beclin-1 expression activated autophagy, it is likely that induction of the autophagic pathway would serve to inhibit apoptosis and preserve the life of the terminally differentiated chondrocyte. We conclude that PIM-2 regulates a new intermediate stage in the differentiation pathway, the induction of autophagy.     

The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.     

Human CD180 Transmits Signals via the PIM-1L Kinase

Toll-like receptors (TLRs) are important sensors of the innate immune system that recognize conserved structural motifs and activate cells via a downstream signaling cascade. The CD180/MD1 molecular complex is an unusual member of the TLR family, since it lacks the components that are normally required for signal transduction by other TLRs. Therefore the CD180/MD 1 complex has been considered of being incapable of independently initiating cellular signals. Using chemogenetic approaches we identified specifically the membrane bound long form of PIM-1 kinase, PIM-1L as the mediator of CD180-dependent signaling. A dominant negative isoform of PIM-1L, but not of other PIM kinases, inhibited signaling elicited by cross-linking of CD180, and this effect was phenocopied by PIM inhibitors. PIM-1L was directed to the cell membrane by its N-terminal extension, where it colocalized and physically associated with CD180. Triggering CD180 also induced increased phosphorylation of the anti-apoptotic protein BAD in a PIM kinase-dependent fashion. Also in primary human B cells, which are the main cells expressing CD180 in man, cross-linking of CD180 by monoclonal antibodies stimulated cell survival and proliferation that was abrogated by specific inhibitors. By associating with PIM-1L, CD180 can thus obtain autonomous signaling capabilities, and this complex is then channeling inflammatory signals into B cell survival programs. Pharmacological inhibition of PIM-1 should therefore provide novel therapeutic options in diseases that respond to innate immune stimulation with subsequently increased B cell activity, such as lupus erythematosus or myasthenia gravis.     

The serine/threonine kinase Pim-1

The human pim-1 gene encodes a serine/threonine kinase, which belongs to the group of calcium/calmodulin-regulated kinases (CAMK). It contains a characteristic kinase domain, a so-called ATP anchor and an active site. In mouse and human, two Pim-1 proteins are produced from the same gene by using an alternative upstream CUG initiation codon, a 44 kD and another, shorter 34 kD form that both contain the kinase domain. Expression of Pim-1 is widespread and ranges from the hematopoietic and lymphoid system to prostate, testis and oral epithelial cells. Two other proteins with significant sequence similarities exist, Pim-2 and Pim-3; both are also serine/threonine kinases and have largely overlapping functions. Pim-1 is able to phosphorylate different targets, most of which are involved in cell cycle progression or apoptosis. Pim-1 expression can be induced by several external stimuli in particular by a number of cytokines relevant in the immune system, which led to the labeling of Pim-1 as a "booster" for the immune response.     

Raf-1 and B-Raf promote protein kinase C theta interaction with BAD

PKCtheta regulates the proliferation, survival and differentiation of T-cells. Here we show that PKCtheta interacts with Raf-1 and B-Raf kinases. Raf-1 enhanced the kinase activity of associated PKCtheta, while PKCtheta reduced the catalytic activity of associated Raf-1. In contrast, B-Raf binding did not affect PKCtheta kinase activity, and PKCtheta did not change B-Raf activity. Coexpression of mutationally activated Raf-1 in cells enhanced the phosphorylation of T538 in the PKCtheta activation loop. PKCtheta and Raf cooperated in terms of binding to BAD, a pro-apoptotic Bcl-2 family protein that is inactivated by phosphorylation. While neither Raf-1 nor B-Raf could phosphorylate BAD, they enhanced the ability of PKCtheta to interact with BAD and to phosphorylate BAD in vitro and in vivo, suggesting a new role for Raf proteins in T-cells by targeting PKCtheta to interact with and phosphorylate BAD.     

The regulation of p53, p38 MAPK,JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.     

The role of PIM kinases in human and mouse CD4+ T cell activation and inflammatory bowel disease

PIM kinases are a family of three serine/threonine kinases expressed following T cell activation. Using potent selective small molecule antagonists of PIM-1/3 kinases, we demonstrate a potential role for these enzymes in naïve and effector CD4+ T cell activation. PIM-1/3 inhibition prevented CD4+ T cell proliferation by inducing a G0/G1 cell cycle arrest without affecting cellular survival. In the absence of PIM-1/3 kinase activity, naïve CD4+ T cells failed to fully differentiate into effector cells both in vitro and in vivo. Therapeutic dosing of a PIM-1/3 inhibitor was efficacious in a CD4+ T cell-mediated model of inflammatory bowel disease suggesting that PIM-1 and PIM-3 kinase activity contributes to sustained disease severity. These results demonstrate that PIM-1/3 kinases have an important role in CD4+ T cell responses and inhibition of this activity may provide a therapeutic benefit in T cell-mediated diseases.     

Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival

Phosphorylation of BAD, a pro-apoptotic member of the Bcl-2 protein family, on either Ser112 or Ser136 is thought to be necessary and sufficient for growth factors to promote cell survival. Here we report that Ser155, a site phosphorylated by protein kinase A (PKA), also contributes to cell survival. Ser112 is thought to be the critical PKA target, but we found that BAD fusion proteins containing Ala at Ser112 (S112A) or Ser136 (S136A) or at both positions (S112/136A) were still heavily phosphorylated by PKA in an in vitro kinase assay. BAD became insensitive to phosphorylation by PKA only when both Ser112 and Ser136, or all three serines (S112/136/155) were mutated to alanine. In HEK293 cells, BAD fusion proteins mutated at Ser155 were refractory to phosphorylation induced by elevation of cyclic AMP(cAMP) levels. Phosphorylation of the S112/136A mutant was >90% inhibited by H89, a PKA inhibitor. The S155A mutant induced more apoptosis than the wild-type protein in serum-maintained CHO-K1 cells, and apoptosis induced by the S112/136A mutant was potentiated by serum withdrawal. These data suggest that Ser155 is a major site of phosphorylation by PKA and serum-induced kinases. Like Ser112 and Ser136, phosphorylation of Ser155 contributes to the cancellation of the pro-apoptotic function of BAD.     

The PIM-2 Kinase Is an Essential Component of the Ultraviolet Damage Response That Acts Upstream to E2F-1 and ATM

The oncogenic nature ascribed to the PIM-2 kinase relies mostly on phosphorylation of substrates that act as pro-survival/anti-apoptotic factors. Nevertheless, pro-survival effects can also result from activating DNA repair mechanisms following damage. In this study, we addressed the possibility that PIM-2 plays a role in the cellular response to UV damage, an issue that has never been addressed before. We found that in U2OS cells, PIM-2 expression and activity increased upon exposure to UVC radiation (2–50 mJ/cm²), and Pim-2-silenced cells were significantly more sensitive to UV radiation. Overexpression of PIM-2 accelerated removal of UV-induced DNA lesions over time, reduced γH2AX accumulation in damaged cells, and rendered these cells significantly more viable following UV radiation. The protective effect of PIM-2 was mediated by increased E2F-1 and activated ATM levels. Silencing E2F-1 reduced the protective effect of PIM-2, whereas inhibiting ATM activity abrogated this protective effect, irrespective of E2F-1 levels. The results obtained in this study place PIM-2 upstream to E2F-1 and ATM in the UV-induced DNA damage response.     

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.     

CaMKII-γ mediates phosphorylation of BAD at Ser170 to regulate cytokine-dependent survival and proliferation

Phosphorylation of the BH3 (Bcl-2 homology domain 3)-only protein BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) can either directly disrupt its association with the pro-survival proteins Bcl-X(L) and/or Bcl-2, or cause association of BAD with 14-3-3 proteins. In the present study, we further characterize phosphorylation of BAD at Ser170, a unique site with unclear function. We provide further evidence that mutation of Ser170 to a phospho-mimetic aspartic acid residue (S170D) can have a profound inhibitory effect on the pro-apoptosis function of BAD. Furthermore, mutated BAD with an alanine substitution inhibited cell proliferation, slowing progression specifically through S-phase. We identify the kinase responsible for phosphorylation at this site as CaMKII-γ (γ isoform of Ca2+/calmodulin-dependent kinase II), but not the other three isoforms of CaMKII, revealing an extraordinary specificity among these closely related kinases. Furthermore, cytokine treatment increased BAD-Ser170-directed CaMKII-γ activity and phosphorylation of CaMKII-γ at an activating site, and CaMKII activity directed to the BAD-Ser170 site was elevated during S-phase. Treating cells with a selective inhibitor of CaMKII caused apoptosis in cells expressing BAD, but not in cells expressing the BAD-S170D mutant. The present study provides support for BAD-Ser170 phosphorylation playing a key role not only in regulating BAD's pro-apoptotic activity, but also in cell proliferation.     

Regulation of BAD protein by PKA, PKCdelta and phosphatases in adult rat cardiac myocytes subjected to oxidative stress

H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases.     

Protein kinase CK2 phosphorylates BAD at threonine-117

Reversible phosphorylation of the 22 kDa BAD protein is crucial for cell survival. Five phosphorylation sites, all serines, had been identified. Here we report on number six. It is threonine-117 phosphorylated by the constitutively active kinase, CK2. Phosphoamino acid analysis and phospho-specific antibodies confirmed Thr117 as additional phosphorylation site. Immunoprecipitation furthermore revealed that BAD is phosphorylated at Thr117 in cultured cortical neurons. PP1, PP2A and PP2C dephosphorylated BAD at Thr117, but PP2B did not. The discovery of the constitutively active CK2 phosphorylating BAD is shedding an unexpected light in the otherwise strictly signal-regulated phosphorylation events on BAD.     

The Pim family of protein kinases: structure, functions and roles in hematopoietic malignancies

Phosphorylation is the universal regulatory mechanism in key physiological processes such as development, cell differentiation, proliferation, survival and malignant transformation. In this review we analyze serine/threonine protein kinases of the Pim (proviral integration of Moloney virus) family that have been initially discovered in experimental lymphomas. We provide data on gene structure, evolution, functions and substrates of Pim protein kinases. Focusing on Pim-1 as the major isoform, we analyze its role in the biology of hematopoietic malignancies. Pim-1 is a pro-proliferative and pro-survival protein kinase. It is constitutively active due to autophosphorylation, and its downstream partners positively regulate the cell cycle. Pim-1 cooperates with c-Myc oncoprotein in leukemogenesis; furthermore, Pim-1, like the Akt protein kinase, prevents cell death. Thus, Pim kinases are regarded as new therapeutic targets. Finally, we present an original test system f or screening of Pim inhibitors. In this test system the growth of a genetically engineered Escherichia coli strain in the presence of kanamycin is dependent on the phosphorylation of aminoglycoside-3' phosphotransferase VIII by Pim-1: pharmacological inhibition of this phosphorylation increases the bacterial cell lysis.     

Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.     

The protective role of a small GTPase RhoE against UVB-induced DNA damage in keratinocytes

RhoE, a p53 target gene, was identified as a critical factor for the survival of human keratinocytes in response to UVB. The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton, acting as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho family proteins, RhoE (also known as Rnd3) is GTPase-deficient and thus expected to be constitutively active. In this study, we investigated the response of cultured human keratinocyte cells to UVB irradiation. RhoE protein levels increase upon exposure to UVB, and ablation of RhoE induction through small interfering RNA resulted in a significant increase in apoptosis and a reduction in the levels of the pro-survival targets p21, Cox-2, and cyclin D1, as well as an increase of reactive oxygen species levels when compared with control cells. These data indicate that RhoE is a pro-survival factor acting upstream of p38, JNK, p21, and cyclin D1. HaCat cells expressing small interfering RNA to p53 indicate that RhoE functions independently of its known associates, p53 and Rho-associated kinase I (ROCK I). Targeted expression of RhoE in epidermis using skin-specific transgenic mouse model resulted in a significant reduction in the number of apoptotic cells following UVB irradiation. Thus, RhoE induction counteracts UVB-induced apoptosis and may serve as a novel target for the prevention of UVB-induced photodamage regardless of p53 status.     

Pro-survival effects of 17β-estradiol on osteocytes are mediated by nitric oxide/cGMP via differential actions of cGMP-dependent protein kinases I and II

Estrogens promote bone health in part by increasing osteocyte survival, an effect that requires activation of the protein kinases Akt and ERK1/2, but the molecular mechanisms involved are only partly understood. Because estrogens increase nitric oxide (NO) synthesis and NO can have anti-apoptotic effects, we examined the role of NO/cGMP signaling in estrogen regulation of osteocyte survival. Etoposide-induced death of MLO-Y4 osteocyte-like cells, assessed by trypan blue staining, caspase-3 cleavage, and TUNEL assays, was completely prevented when cells were pre-treated with 17β-estradiol. This protective effect was mimicked when cells were pre-treated with a membrane-permeable cGMP analog and blocked by pharmacological inhibitors of NO synthase, soluble guanylate cyclase, or cGMP-dependent protein kinases (PKGs), supporting a requirement for NO/cGMP/PKG signaling downstream of 17β-estradiol. siRNA-mediated knockdown and viral reconstitution of individual PKG isoforms demonstrated that the anti-apoptotic effects of estradiol and cGMP were mediated by PKG Iα and PKG II. Akt and ERK1/2 activation by 17β-estradiol required PKG II, and cGMP mimicked the effects of estradiol on Akt and ERK, including induction of ERK nuclear translocation. cGMP induced BAD phosphorylation on several sites, and experiments with phosphorylation-deficient BAD mutants demonstrated that the anti-apoptotic effects of cGMP and 17β-estradiol required BAD phosphorylation on Ser(136) and Ser(155); these sites were targeted by Akt and PKG I, respectively, and regulate BAD interaction with Bcl-2. In conclusion, 17β-estradiol protects osteocytes against apoptosis by activating the NO/cGMP/PKG cascade; PKG II is required for estradiol-induced activation of ERK and Akt, and PKG Iα contributes to pro-survival signaling by directly phosphorylating BAD.     

Activation of a histone H1 kinase by tyrosine phosphorylation in v-src-transformed fibroblasts.

Using anti-phosphotyrosine immunoaffinity chromatography, we have searched for serine/threonine kinases that are directly regulated by tyrosine phosphorylation in v-src-transformed rat 3Y1 fibroblasts. Tyrosine phosphoprotein preparations from v-src-transformed cells contain a kinase activity that phosphorylates histone H1 in vitro on serine residues and this activity is present at a 20-fold greater level than that in parental cell preparations. This activity elutes from a MonoQ FPLC column as a single peak and gel filtration chromatography suggests that the kinase has a molecular mass of approximately 55 kDa. Tyrosine phosphatase treatment inactivates the histone H1 kinase and this result indicates that the specific activity of the kinase is regulated by tyrosine phosphorylation. Experiments with cells transformed with a temperature-sensitive mutant of the v-src oncogene demonstrate that the tyrosine phosphorylation of the histone H1 kinase is an early event in v-src transformation. The kinase is distinct from known cdc2 family members that contain the PSTAIR motif, because the kinase can be separated almost completely from these proteins by immunoprecipitation with an antibody against p34cdc2. The profile of antibody reactivity and sensitivity to modulators of protein kinases suggests that this activity is distinct from known second messenger-regulated kinases and from previously characterized MAP kinases.