USP49 negatively regulates tumorigenesis and chemoresistance through FKBP51‐AKT signaling

The AKT pathway is a fundamental signaling pathway that mediates multiple cellular processes, such as cell proliferation and survival, angiogenesis, and glucose metabolism. We recently reported that the immunophilin FKBP51 is a scaffolding protein that can enhance PHLPP‐AKT interaction and facilitate PHLPP‐mediated dephosphorylation of AKT at Ser473, negatively regulating AKT activation. However, the regulation of FKBP51‐PHLPP‐AKT pathway remains unclear. Here we report that a deubiquitinase, USP49, is a new regulator of the AKT pathway. Mechanistically, USP49 deubiquitinates and stabilizes FKBP51, which in turn enhances PHLPP's capability to dephosphorylate AKT. Furthermore, USP49 inhibited pancreatic cancer cell proliferation and enhanced cellular response to gemcitabine in a FKBP51‐AKT‐dependent manner. Clinically, decreased expression of USP49 in patients with pancreatic cancer was associated with decreased FKBP51 expression and increased AKT phosphorylation. Overall, our findings establish USP49 as a novel regulator of AKT pathway with a critical role in tumorigenesis and chemo‐response in pancreatic cancer.     

Diacylglycerol Kinase δ Modulates Akt Phosphorylation through Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase 2 (PHLPP2)

Discovering proteins that modulate Akt signaling has become a critical task, given the oncogenic role of Akt in a wide variety of cancers. We have discovered a novel diacylglycerol signaling pathway that promotes dephosphorylation of Akt. This pathway is regulated by diacylglycerol kinase δ (DGKδ). In DGKδ-deficient cells, we found reduced Akt phosphorylation downstream of three receptor tyrosine kinases. Phosphorylation upstream of Akt was not affected. Our data indicate that PKCα, which is excessively active in DGKδ-deficient cells, promotes dephosphorylation of Akt through pleckstrin homology domain leucine-rich repeats protein phosphatase (PHLPP) 2. Depletion of either PKCα or PHLPP2 rescued Akt phosphorylation in DGKδ-deficient cells. In contrast, depletion of PHLPP1, another Akt phosphatase, failed to rescue Akt phosphorylation. Other PHLPP substrates were not affected by DGKδ deficiency, suggesting mechanisms allowing specific modulation of Akt dephosphorylation. We found that β-arrestin 1 acted as a scaffold for PHLPP2 and Akt1, providing a mechanism for specificity. Because of its ability to reduce Akt phosphorylation, we tested whether depletion of DGKδ could attenuate tumorigenic properties of cultured cells and found that DGKδ deficiency reduced cell proliferation and migration and enhanced apoptosis. We have, thus, discovered a novel pathway in which diacylglycerol signaling negatively regulates Akt activity. Our collective data indicate that DGKδ is a pertinent cancer target, and our studies could lay the groundwork for development of novel cancer therapeutics.     

InterAKTions with FKBPs - Mutational and Pharmacological Exploration

The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases. In pancreatic cancer cell lines, FKBP51 was shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of the kinase Akt, which was associated with reduced chemoresistance. Here we show that in addition to FKBP51 several other members of the FKBP family bind directly to Akt. FKBP51 can also form complexes with other AGC kinases and mapping studies revealed that FKBP51 interacts with Akt via multiple domains independent of their activation or phosphorylation status. The FKBP51-Akt1 interaction was not affected by FK506 analogs or Akt active site inhibitors, but was abolished by the allosteric Akt inhibitor VIII. None of the FKBP51 inhibitors affected AktS473 phosphorylation or downstream targets of Akt. In summary, we show that FKBP51 binds to Akt directly as well as via Hsp90. The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51. Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.     

Relationship between polycomb‐group protein BMI‐1 and phosphatases regulating AKT phosphorylation level in endometrial cancer

The PI3K/AKT pathway is frequently activated in endometrial carcinoma. BMI‐1 (B‐lymphoma Mo‐MLV insertion region 1) protein affects expression of PTEN (phosphatase and tensin homolog) in some cancers, but its significance for endometrial tumorigenesis is not known. The objective of this study was to determine the relationship between BMI‐1 and expression of factors affecting AKT (protein kinase B) phosphorylation level in endometrial cancer. The expression of proteins and mRNAs was investigated in endometrial cancer specimens and samples of non‐neoplastic endometrial tissue by Western blot and RT‐PCR, respectively. The impact of BMI‐1 down‐regulation on AKT phosphorylation and expression of genes coding for several phosphatases were studied in HEC1A cells. The results showed that BMI‐1 depletion caused increase in PHLPP1 and PHLPP2 (PH domain and leucine‐rich repeat protein phosphatases 1/2) expression and decrease in phospho‐AKT (pAKT) level. In more advanced tumours with higher metastatic potential, the expression of BMI‐1 was lower compared to tumours less advanced and without lymph node metastasis. There were significant inverse correlations between BMI‐1 and PHLPPs, especially PHLPP1 in normal endometrial samples. The inverse correlation between BMI‐1 and PHLPP1/PHLPP2 expression was observed in PTEN positive but not PTEN negative cancers. Low PHLPP2 expression in tumours predicted poorer overall survival. BMI‐1 impacts on AKT phosphorylation level in endometrial cells by regulation of PHLPP expression.     

Phosphatase PHLPP2 regulates the cellular response to metabolic stress through AMPK

PHLPP2 is a member of the PHLPP family of phosphatases, known to suppress cell growth by inhibiting proliferation or promoting apoptosis. Oncogenic kinases Akt, S6K, and PKC, and pro-apoptotic kinase Mst1, have been recognized as functional targets of the PHLPP family. However, we observed that, in T-leukemia cells subjected to metabolic stress from glucose limitation, PHLPP2 specifically targets the energy-sensing AMP-activated protein kinase, pAMPK, rather than Akt or S6K. PHLPP2 dephosphorylates pAMPK in several other human cancer cells as well. PHLPP2 and pAMPK interact with each other, and the pleckstrin homology (PH) domain on PHLPP2 is required for their interaction, for dephosphorylating and inactivating AMPK, and for the apoptotic response of the leukemia cells to glucose limitation. Silencing PHLPP2 protein expression prolongs the survival of leukemia cells subjected to severe glucose limitation by promoting a switch to AMPK-mediated fatty acid oxidation for energy generation. Thus, this study reveals a novel role for PHLPP2 in suppressing a survival response mediated through AMPK signaling. Given the multiple ways in which PHLPP phosphatases act to oppose survival signaling in cancers and the pivotal role played by AMPK in redox homeostasis via glucose and fatty acid metabolism, the revelation that AMPK is a target of PHLPP2 could lead to better therapeutics directed both at cancer and at metabolic diseases.Subject terms: Cancer metabolism, Stress signalling     

Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway,through endoplasmic reticulum stress to induce autophagy

Hepatitis C virus (HCV) is able to induce autophagy via endoplasmic reticulum (ER) stress, but the exact molecular signaling pathway is not well understood. We found that the activity of the mechanistic target of rapamycin complex 1 (MTORC1) was inhibited in Huh7 cells either harboring HCV-N (genotype 1b) full-genomic replicon or infected with JFH1 (genotype 2a) virus, which led to the activation of UNC-51-like kinase 1 (ULK1) and thus to autophagy. We then analyzed activity upstream of MTORC1, and found that both protein kinase, AMP-activated, α (PRKAA, including PRKAA1 and PRKAA2, also known as AMP-activated protein kinase, AMPKα) and AKT (refers to pan AKT, including three isoforms of AKT1-3, also known as protein kinase B, PKB) were inhibited by HCV infection. The inhibition of the AKT-TSC-MTORC1 pathway contributed to upregulating autophagy, but inhibition of PRKAA downregulated autophagy. The net effect on autophagy was from AKT, which overrode the inhibition effect from PRKAA. It was further found that HCV-induced ER stress was responsible for the inhibition of the AKT pathway. Metformin, a PRKAA agonist, inhibited HCV replication not only by activating PRKAA as previously reported, but also by activating AKT independently of the autophagy pathway. Taken together, our data suggested HCV inhibited the AKT-TSC-MTORC1 pathway via ER stress, resulting in autophagy, which may contribute to the establishment of the HCV-induced autophagy.     

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.     

Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylinositol 3-kinase/AKT cascades

Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.     

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex.SummaryIn this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.     

Network Reconstruction and Significant Pathway Extraction Using Phosphoproteomic Data from Cancer Cells

Protein phosphorylation acts as an efficient switch controlling deregulated key signaling pathway in cancer. Computational biology aims to address the complexity of reconstructed networks but overrepresents well‐known proteins and lacks information on less‐studied proteins. A bioinformatic tool to reconstruct and select relatively small networks that connect signaling proteins to their targets in specific contexts is developed. It enables to propose and validate new signaling axes of the Syk kinase. To validate the potency of the tool, it is applied to two phosphoproteomic studies on oncogenic mutants of the well‐known phosphatidyl‐inositol 3‐kinase (PIK3CA) and the unfamiliar Src‐related tyrosine kinase lacking C‐terminal regulatory tyrosine and N‐terminal myristoylation sites (SRMS) kinase. By combining network reconstruction and signal propagation, comprehensive signaling networks from large‐scale experimental data are built and multiple molecular paths from these kinases to their targets are extracted. Specific paths from two distinct PIK3CA mutants are retrieved, and their differential impact on the HER3 receptor kinase is explained. In addition, to address the missing connectivities of the SRMS kinase to its targets in interaction pathway databases, phospho‐tyrosine and phospho‐serine/threonine proteomic data are integrated. The resulting SRMS‐signaling network comprises casein kinase 2, thereby validating its currently suggested role downstream of SRMS. The computational pipeline is publicly available, and contains a user‐friendly graphical interface ( http://doi.org/10.5281/zenodo.3333687 ).     

The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration

Disruption of autophagy plays an import role in neurodegenerative disorders, where deficient elimination of abnormal and toxic protein aggregates promotes cellular stress, failure and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. Upstream of mTOR the survival PI3K/AKT pathway modulates mTOR activity that is also altered in neurodegenerative diseases of Alzheimer and Parkinson. Nevertheless, the interplay between the PI3K/AKT/mTOR pathway and the autophagic process is complex and a more detailed examination of tissue from patients suffering neurodegenerative diseases and of animal and cellular models is needed. In the present work we review the recent findings on the role of the PI3K/AKT/mTOR pathway in the modulation of the autophagic process in neuronal protection.     

PI3K-independent AKT activation in cancers: a treasure trove for novel therapeutics

AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors.     

Abl kinases are required for invadopodia formation and chemokine-induced invasion

The Abl tyrosine kinases, Abl and Arg, play a role in the regulation of the actin cytoskeleton by modulating cell-cell adhesion and cell motility. Deregulation of both the actin cytoskeleton and Abl kinases have been implicated in cancers. Abl kinase activity is elevated in a number of metastatic cancers and these kinases are activated downstream of several oncogenic growth factor receptor signaling pathways. However, the role of Abl kinases in regulation of the actin cytoskeleton during tumor progression and invasion remains elusive. Here we identify the Abl kinases as essential regulators of invadopodia assembly and function. We show that Abl kinases are activated downstream of the chemokine receptor, CXCR4, and are required for cancer cell invasion and matrix degradation induced by SDF1α, serum growth factors, and activated Src kinase. Moreover, Abl kinases are readily detected at invadopodia assembly sites and their inhibition prevents the assembly of actin and cortactin into organized invadopodia structures. We show that active Abl kinases form complexes with membrane type-1 matrix metalloproteinase (MT1-MMP), a critical invadopodia component required for matrix degradation. Further, loss of Abl kinase signaling induces internalization of MT1-MMP from the cell surface, promotes its accumulation in the perinuclear compartment and inhibits MT1-MMP tyrosine phosphorylation. Our findings reveal that Abl kinase signaling plays a critical role in invadopodia formation and function, and have far-reaching implications for the treatment of metastatic carcinomas.     

FKBP51 employs both scaffold and isomerase functions to promote NF-κB activation in melanoma

Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-κB constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-κB activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKKγ to the IKK catalytic subunits, IKK-α and -β, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKKγ, whereas only the TPR domain is involved in interactions with IKKα and β. Collectively, these results suggest that FKBP51 promotes NF-κB activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms

Akt/protein kinase B controls cell growth, proliferation, and survival. We recently discovered a novel phosphatase PHLPP, for PH domain leucine-rich repeat protein phosphatase, which terminates Akt signaling by directly dephosphorylating and inactivating Akt. Here we describe a second family member, PHLPP2, which also inactivates Akt, inhibits cell-cycle progression, and promotes apoptosis. These phosphatases control the amplitude of Akt signaling: depletion of either isoform increases the magnitude of agonist-evoked Akt phosphorylation by almost two orders of magnitude. Although PHLPP1 and PHLPP2 both dephosphorylate the same residue (hydrophobic phosphorylation motif) on Akt, they differentially terminate Akt signaling by regulating distinct Akt isoforms. Knockdown studies reveal that PHLPP1 specifically modulates the phosphorylation of HDM2 and GSK-3alpha through Akt2, whereas PHLPP2 specifically modulates the phosphorylation of p27 through Akt3. Our data unveil a mechanism to selectively terminate Akt-signaling pathways through the differential inactivation of specific Akt isoforms by specific PHLPP isoforms.     

Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34⁺ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.     

Blockade of the ERK or PI3K-Akt signaling pathway enhances the cytotoxicity of histone deacetylase inhibitors in tumor cells resistant to gefitinib or imatinib

Deregulated activation of protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) and Abl, is associated with human cancers including non-small cell lung cancer (NSCLC) and chronic myeloid leukemia (CML). Although inhibitors of such activated kinases have proved to be of therapeutic benefit in individuals with NSCLC or CML, some patients manifest intrinsic or acquired resistance to these drugs. We now show that, whereas blockade of either the extracellular signal-regulated kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3K)-Akt pathway alone induced only a low level of cell death, it markedly sensitized NSCLC or CML cells to the induction of apoptosis by histone deacetylase (HDAC) inhibitors. Such enhanced cell death induced by the respective drug combinations was apparent even in NSCLC or CML cells exhibiting resistance to EGFR or Abl tyrosine kinase inhibitors, respectively. Co-administration of a cytostatic signaling pathway inhibitor may contribute to the development of safer anticancer strategies by lowering the required dose of cytotoxic HDAC inhibitors for a variety of cancers.     

Novel effects of sphingosylphosphorylcholine on the apoptosis of breast cancer via autophagy/AKT/p38 and JNK signaling

Sphingosylphosphorylcholine (SPC), an important lipid mediator in blood, inhibits the proliferation and migration of various cancer cells. However, its effect as a cell-specific sphingolipid in breast cancer cells is still unknown. Here, we showed that SPC promoted autophagy and apoptosis in triple-negative breast cancer MDA-MB-231 cells. Autophagy worked as a negative regulator of apoptosis-induced by SPC. Mechanistically, SPC mediated apoptosis via activating c-Jun N-terminal kinase (JNK). Meanwhile, p38MAPK (p38) and protein kinase B (PKB or AKT) signaling pathways were also activated to inhibit apoptosis, suggesting that SPC could evoke multiple signaling pathways to modulate cell apoptosis. In addition, the crosstalk between autophagy, p38, AKT and JNK is that autophagy, p38, and AKT attenuated the JNK. AKT and p38 were in the downstream of autophagy, which is autophagy/AKT/p38 signaling evoked by SPC to antagonize JNK signaling and subsequent apoptosis. Although the pathways that antagonize apoptosis were evoked, the cells eventually reached apoptosis by SPC. Therefore, the combination with pharmacological autophagy inhibitors would be a more effective therapeutic strategy for eliminating breast cancer cells by SPC.