NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling

ARK5 is a tumor progression-associated factor that is directly phosphorylated by AKT at serine 600 in the regulatory domain, but phosphorylation at the conserved threonine residue on the active T loop has been found to be required for its full activation. In this study, we identified serine/threonine protein kinase NDR2 as a protein kinase that phosphorylates and activates ARK5 during insulin-like growth factor (IGF)-1 signaling. Upon stimulation with IGF-1, NDR2 was found to directly phosphorylate the conserved threonine 211 on the active T loop of ARK5 and to promote cell survival and invasion of colorectal cancer cell lines through ARK5. During IGF-1 signaling, phosphorylation at three residues (threonine 75, serine 282, and threonine 442) was also found to be required for NDR2 activation. Among these three residues, phosphorylation of serine 282 seemed to be the most important for NDR2 activation (the same as for the mouse homologue) because its aspartic acid-converted mutant (NDR2/S282D) induced ARK5-mediated cell survival and invasion activities even in the absence of IGF-1. As in the mouse homologue, threonine 75 in NDR2 was required for interaction with S100B, and binding was in a calcium ion- and phospholipase C-gamma-dependent manner. We also found that PDK-1 plays an important role in NDR2 activation especially in the phosphorylation of threonine 442. Based on the results of this study, we report here that NDR2 is an upstream kinase of ARK5 that plays an essential role in tumor progression through ARK5.     

Gβγ interacts with mTOR and promotes its activation

Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ₄. Both, mTORC1 and mTORC2 complexes interact with Gβ₁γ₂ which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.     

A critical role for AKT activation in protecting cells from ionizing radiation-induced apoptosis and the regulation of acinus gene expression

Although AKT activation leads to the activation of various pathways related to cell survival, the roles of AKT in modulating cellular responses induced by ionizing radiation in normal human cells remain unclear. Here we show that low-dose radiation of 0.05 Gy did not affect cell death, but high-dose radiation (> 0.2 Gy) induced apoptosis through the activation of caspases and acinus cleavage. Ionizing radiation induced acinus phosphorylation via AKT activation. Thus, we examined the effect of AKT activation on radiation-induced cell death using CCD-18Lu cells transduced with a retroviral vector expressing constitutively active AKT (CA-AKT). The overexpression of CA-AKT rendered the cells resistant to ionizing radiation and prevented the proteolytic cleavage of acinus via phosphorylation. In addition, overexpression of CA-AKT resulted in the upregulation of acinus expression by activation of the NF-κB pathway. On the other hand, suppression of endogenous AKT expression by siRNA resulted in the reduction of acinus expression and enhanced the radiation-induced apoptosis in both CCD-18Lu and IM-9 cells. Our results suggest that AKT activation inhibits cell death during radiation-induced apoptosis through the regulation of phosphorylation and expression of acinus. The AKT/NF-κB/acinus pathway functions as one of the important regulatory mechanisms required for modulating ionizing radiation sensitivity.     

CaM kinase control of AKT and LNCaP cell survival

AKT and its substrate BAD have been shown to promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. The LNCaP prostate cancer cells express the carbachol-sensitive M(3) -subtype of G protein-coupled receptors that cause increases in intracellular calcium and activate the family of Ca(2+) /calmodulin-dependent protein kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), phosphorylates several substrates including AKT on threonine 308. AKT phosphorylation and activation enhances cell survival through phosphorylation of BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT and BAD in LNCaP prostate cancer cells and evaluate whether CaM KK may be mediating carbachol's activation of AKT and cell survival. Our results suggest that carbachol treatment of LNCaP cells promoted cell survival through CaM KK and its phosphorylation of AKT. The bacterial toxin anisomycin triggered caspase-3 activation in LNCaP cells that was blocked by carbachol in a CaM KK- and AKT-dependent manner. AKT and BAD phosphorylation were blocked by the selective CaM KK inhibitor, STO-609, as well as siRNA directed against CaM KK. BAD phosphorylation was also blocked by treating cells with the AKT inhibitor, AKT-X, as well as siRNA to AKT. Additionally, epinephrine promoted LNCaP cell survival through activation of AKT that was insensitive to STO-609. Taken together these data suggest a survival role for CaM KK operating through AKT and BAD in LNCaP prostate cancer cells.     

Negative regulation of mixed lineage kinase 3 by protein kinase B/AKT leads to cell survival

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKBalpha. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.     

Protein kinase C phosphorylates the EphA2 receptor on serine 892 in the regulatory linker connecting the kinase and SAM domains

The EphA2 receptor tyrosine kinase signals through two distinct mechanisms, one regulated by tyrosine phosphorylation and the other by serine/threonine phosphorylation. Serine 892 (S892) is one of the major serine/threonine phosphorylation sites in EphA2, but little is known about its regulation and function. S892 is located in the linker connecting the EphA2 kinase and SAM domains, and is part of a cluster of five phosphorylated residues that includes the well characterized S897. EphA2 can be phosphorylated on S897 by the RSK, AKT and PKA kinases to promote a non-canonical form of signaling that plays an important role in cancer malignancy. Here we show that the Protein Kinase C (PKC) family phosphorylates the EphA2 S892 motif in vitro and in cells. By using a newly developed phosphospecific antibody, we detected EphA2 S892 phosphorylation in a variety of cell lines. As expected for a PKC target site, the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) increases S892 phosphorylation whereas the broad-spectrum PKC inhibitor Go 6983 inhibits both basal and TPA-induced S892 phosphorylation. Besides phosphorylating S892, PKC can also increase EphA2 phosphorylation on S897 through the MEK kinase, which regulates the ERK-RSK signaling axis. We also found that S892 and S897 phosphorylation induced by PKC activation can be downregulated by ephrin ligand-induced EphA2 canonical signaling. Our data reveal that the PKC family contributes to the phosphorylation cluster in the EphA2 kinase-SAM linker, which regulates EphA2 non-canonical signaling and cancer malignancy.     

Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance

The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.     

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.     

The Effects of Low-dose Ionizing Radiation in the Activated Rat Basophilic Leukemia (RBL-2H3) Mast Cells

Mast cells play important roles in many biological responses, such as those during allergic diseases and inflammatory disorders. Although laser and UV irradiation have immunosuppressive effects on inflammatory diseases by suppressing mast cells, little is known about the effects of γ-ionizing radiation on mast cells. In this study, we investigated the effects of γ-ionizing radiation on RBL-2H3 cells, a convenient model system for studying regulated secretion by mast cells. Low-dose radiation (<0.1 gray (Gy)) did not induce cell death, but high-dose radiation (>0.5 Gy) induced apoptosis. Low-dose ionizing radiation significantly suppressed the release of mediators (histamine, β-hexosaminidase, IL-4, and tumor necrosis factor-α) from immunoglobulin E (IgE)-sensitized RBL-2H3 cells. To determine the mechanism of mediator release inhibition by ionizing radiation, we examined the activation of intracellular signaling molecules such as Lyn, Syk, phospholipase Cγ, PKCs, and MAPK, and intracellular free calcium concentrations ([Ca(2+)](i)). The phosphorylation of signaling molecules following stimulation of high-affinity IgE receptor I (FcεRI) was specifically inhibited by low-dose ionizing radiation (0.01 Gy). These results were due to the suppression of FcεRI expression by the low-dose ionizing radiation. Therefore, low-dose ionizing radiation (0.01 Gy) may function as a novel inhibitor of mast cell activation.     

EGF receptor phosphorylation is affected by ionizing radiation

Eukaryotic cells respond to ionizing radiation with cell cycle arrest, activation of DNA repair mechanisms, and lethality. However, little is known about the molecular mechanisms that constitute these responses. Here we report that ionizing radiation enhances epidermal growth factor (EGF) receptor tyrosine phosphorylation in intact cells as well as in isolated membranes of A431 cells. Phosphoamino acid analysis revealed that ionizing radiation preferentially enhances tyrosine phosphorylation, while EGF enhances the phosphorylation of all three phosphoamino acids (serine, threonine and tyrosine) of the EGF receptor. In addition, radiation reduces the turnover rate of the EGF receptor, while EGF increases the rate of the receptor turnover and down-regulation. Moreover, the confined radiation-induced phosphorylation of tyrosine residues is inhibited by genistein, indicating that this phosphorylation of EGF receptor is due to protein tyrosine kinase activation. These studies provide novel insights into the capacity of radiation to modulate EGF receptor phosphorylation and function. The radiation-induced elevation in the EGF receptor tyrosine phosphorylation and the receptor's slower rate of turnover are discussed in terms of their possible role in cell growth and apoptosis modulation.     

The CLK family kinases, CLK1 and CLK2, phosphorylate and activate the tyrosine phosphatase, PTP-1B.

The protein-tyrosine phosphatase PTP-1B is an important regulator of intracellular protein tyrosine phosphorylation, and is itself regulated by phosphorylation. We report that PTP-1B and its yeast analog, YPTP, are phosphorylated and activated by members of the CLK family of dual specificity kinases. CLK1 and CLK2 phosphorylation of PTP-1B in vitro activated the phosphatase activity approximately 3-5-fold using either p-nitrophenol phosphate, or tyrosine-phosphorylated myelin basic protein as substrates. Co-expression of CLK1 or CLK2 with PTP-1B in HEK 293 cells led to a 2-fold stimulation of phosphatase activity in vivo. Phosphorylation of PTP-1B at Ser(50) by CLK1 or CLK2 is responsible for its enzymatic activation. These findings suggest that phosphorylation at Ser(50) by serine threonine kinases may regulate the activation of PTP-1B in vivo. We also show that CLK1 and CLK2 phosphorylate and activate the S. cerevisiae PTP-1B family member, YPTP1. CLK1 phosphorylation of YPTP1 led to a 3-fold stimulation of phosphatase activity in vitro. We demonstrate that CLK phosphorylation of Ser(83) on YPTP1 is responsible for the activation of this enzyme. These findings demonstrate that the CLK kinases activate PTP-1B family members, and this phosphatase may be an important cellular target for CLK action.     

Syndecan 4 regulation of PDK1-dependent Akt activation

The phosphatidylinositol 3 kinase (Pi3K)/Akt pathway is a major regulator of cell growth, proliferation, metabolism, survival, and angiogenesis. Despite extensive study, a thorough understanding of the modulation and regulation of this pathway has remained elusive. We have previously demonstrated that syndecan 4 (S4) regulates the intracellular localization of mTORC2, thus altering phosphorylation of Akt at serine473 (Ser473), one of two critical phosphorylation sites essential for the full activation of Akt [1]. Here we report that S4 also regulates the phosphorylation of Akt at threonine308 (Thr308), the second phosphorylation site required for the full Akt activation. A deletion of S4 resulted in lower levels of Thr308 phosphorylation both in vitro and in vivo. Furthermore, a deletion or knockdown of the S4 effector molecule PKCα led to a similar reduction in phosphorylation of Thr308 while overexpression of myristoylated PKCα rescued AktThr308 phosphorylation in endothelial cells lacking S4. Finally, PAK1/2 is also recruited to the rafts by the S4-PKCα complex and is required for AKT activation.     

Feedback loops blockade potentiates apoptosis induction and antitumor activity of a novel AKT inhibitor DC120 in human liver cancer

The serine/threonine kinase AKT is generally accepted as a promising anticancer therapeutic target. However, the relief of feedback inhibition and enhancement of other survival pathways often attenuate the anticancer effects of AKT inhibitors. These compensatory mechanisms are very complicated and remain poorly understood. In the present study, we found a novel 2-pyrimidyl-5-amidothiazole compound, DC120, as an ATP competitive AKT kinase inhibitor that suppressed proliferation and induced apoptosis in liver cancer cells both in vitro and in vivo. DC120 blocked the phosphorylation of downstream molecules in the AKT signal pathway in dose- and time-dependent manners both in vitro and in vivo. However, unexpectedly, DC120 activated mammalian target of rapamycin complex 1 (mTORC1) pathway that was suggested by increased phosphorylation of 70KD ribosomal protein S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The activated mTORC1 signal was because of increase of intracellular Ca²⁺ via Ca²⁺/calmodulin (CaM)/ signaling to human vacuolar protein sorting 34 (hVps34) upon AKT inhibition. Meanwhile, DC120 attenuated the inhibitory effect of AKT on CRAF by decreasing phosphorylation of CRAF at Ser259 and thus activated the mitogen-activated protein kinase (MAPK) pathway. The activation of the mTORC1 and MAPK pathways by DC120 was not mutually dependent, and the combination of DC120 with mTORC1 inhibitor and/or MEK inhibitor induced significant apoptosis and growth inhibition both in vitro and in vivo. Taken together, the combination of AKT, mTORC1 and/or MEK inhibitors would be a promising therapeutic strategy for liver cancer treatment.     

Homeodomain-interacting protein kinase-2 stabilizes p27(kip1) by its phosphorylation at serine 10 and contributes to cell motility

HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53.     

The PEA-15 Protein Regulates Autophagy via Activation of JNK

PEA-15/PED (phosphoprotein enriched in astrocytes 15 kDa/phosphoprotein enriched in diabetes) is a death effector domain-containing protein which is known to modulate apoptotic cell death. The mechanism by which PEA-15 inhibits caspase activation and increases ERK (extracellular-regulated kinase) activity is well characterized. Here, we demonstrate that PEA-15 is not only pivotal in the activation of the ERK pathway but also modulates JNK (c-Jun N-terminal kinase) signaling. Upon overexpression of PEA-15 in malignant glioma cells, JNK is potently activated. The PEA-15-induced JNK activation depends on the phosphorylation of PEA-15 at both phosphorylation sites (serine 104 and serine 116). The activation of JNK is substantially inhibited by siRNA-mediated down-regulation of endogenous PEA-15. Moreover, we demonstrate that glioma cells overexpressing PEA-15 show increased signs of autophagy in response to classical autophagic stimuli such as ionizing irradiation, serum deprivation, or rapamycin treatment. In contrast, the non-phosphorylatable mutants of PEA-15 are not capable of promoting autophagy. The inhibition of JNK abrogates the PEA-15-mediated increase in autophagy. In conclusion, our data show that PEA-15 promotes autophagy in glioma cells in a JNK-dependent manner. This might render glioma cells more resistant to adverse stimuli such as starvation or ionizing irradiation.     

The dual-specificity CLK kinase induces neuronal differentiation of PC12 cells.

CLK is a dual-specificity protein kinase capable of phosphorylating serine, threonine, and tyrosine residues. We have investigated the action of CLK by establishing stable PC12 cell lines capable of inducibly expressing CLK. Expression of CLK in stably transfected PC12 cells mimicked a number of nerve growth factor (NGF)-dependent events, including the morphological differentiation of these cells and the elaboration of neurites. Moreover, CLK expression enhanced the rate of NGF-mediated neurite outgrowth of these cells, indicating that CLK expression and NGF treatment activate similar signal transduction pathways. CLK expression, unlike NGF, was not able to promote PC12 cell survival in serum-free media, demonstrating that CLK only partially recapitulated the actions of NGF on these cells and that the biochemical pathways necessary for morphological differentiation can be stimulated without also stimulating those necessary for survival. Induction of CLK expression also resulted in the selective activation of protein kinases that are components of growth factor-stimulated signal transduction cascades, including ERK1, ERK2, pp90RSK, and S6PKII. Induction of CLK expression, however, did not stimulate pp70S6K or Fos kinase, two NGF-sensitive protein kinases. These data indicate that CLK action mediates the morphological differentiation of these cells through its capacity to independently stimulate signal transduction pathways normally employed by NGF.