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In this issue of Cancer Cell, Ooms and colleagues show that the lipid phosphatase PIPP/INPP5J, frequently inactivated in triple-negative breast cancers, functions as a tumor suppressor by specifically modulating the activity of AKT1 in the context of oncogenic PI3K signaling, leading to inhibition of metastatic dissemination.
Copyright © 2015 Elsevier Inc. All rights reserved.
Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-β, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.
Thirty years ago, glycerolipids captured the attention of biochemical researchers as novel cellular signaling entities. We now recognize that these biomolecules occupy signaling nodes critical to a number of physiological and pathological processes. Thus, glycerolipid-metabolizing enzymes present attractive targets for new therapies. A number of fields-ranging from neuroscience and cancer to diabetes and obesity-have elucidated the signaling properties of glycerolipids. The biochemical literature teems with newly emerging small molecule inhibitors capable of manipulating glycerolipid metabolism and signaling. This ever-expanding pool of chemical modulators appears daunting to those interested in exploiting glycerolipid-signaling pathways in their model system of choice. This review distills the current body of literature surrounding glycerolipid metabolism into a more approachable format, facilitating the application of small molecule inhibitors to novel systems. This article is part of a Special Issue entitled Tools to study lipid functions.
Copyright © 2014 Elsevier B.V. All rights reserved.
Sensitive and specific biomarkers of protein kinase inhibition can be leveraged to accelerate drug development studies in oncology by associating early molecular responses with target inhibition. In this study, we utilized unbiased shotgun phosphotyrosine (pY) proteomics to discover novel biomarkers of response to dasatinib, a small molecule Src-selective inhibitor, in preclinical models of colorectal cancer (CRC). We performed unbiased mass spectrometry shotgun pY proteomics to reveal the pY proteome of cultured HCT-116 colonic carcinoma cells, and then extended this analysis to HCT-116 xenograft tumors to identify pY biomarkers of dasatinib-responsiveness in vivo. Major dasatinib-responsive pY sites in xenograft tumors included sites on delta-type protein kinase C (PKCδ), CUB-domain-containing protein 1 (CDCP1), Type-II SH2-domain-containing inositol 5-phosphatase (SHIP2), and receptor protein-tyrosine phosphatase alpha (RPTPα). The pY313 site PKCδ was further supported as a relevant biomarker of dasatinib-mediated Src inhibition in HCT-116 xenografts by immunohistochemistry and immunoblotting with a phosphospecific antibody. Reduction of PKCδ pY313 was further correlated with dasatinib-mediated inhibition of Src and diminished growth as spheroids of a panel of human CRC cell lines. These studies reveal PKCδ pY313 as a promising readout of Src inhibition in CRC and potentially other solid tumors and may reflect responsiveness to dasatinib in a subset of colorectal cancers.
In cancer, deregulated signaling can produce an invasive cellular phenotype. We modeled the invasive transition as a theoretical switch between two cytoskeletal structures: focal adhesions and extracellular matrix-degrading invadopodia. We constructed molecular interaction networks of each structure and identified upstream regulatory hubs through computational analyses. We compared these regulatory hubs to the status of signaling components from head and neck carcinomas, which led us to analyze phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα). Consistent with previous studies, PI3K activity promoted both the formation and the activity of invadopodia. We found that PI3K induction of invadopodia was increased by overexpression of SH2 (Src homology 2) domain-containing inositol 5'-phosphatase 2 (SHIP2), which converts the phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] that is produced by PI3K activity to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], which is believed to promote invadopodia formation. Knockdown of PKCα had divergent effects on invadopodia formation, depending on the status of PI3K. Loss of PKCα inhibited invadopodia formation in cells with wild-type PI3K pathway status. Conversely, in cells with constitutively active PI3K (through activating PI3K mutants or lacking the endogenous opposing enzyme PTEN), PKCα knockdown increased invadopodia formation. Mechanistic studies revealed a negative feedback loop from PKCα that dampened PI3K activity and invasive behavior in cells with genetic hyperactivation of the PI3K pathway. These studies demonstrated the potential of network modeling as a discovery tool and identified PI3K and PKCα as interacting regulators of invasive behavior.
BACKGROUND - The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS-->pTS-->pTpS-->TpS-->TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC.
METHODOLOGY AND PRINCIPAL FINDINGS - The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP gamma-phosphate. T432 is phosphorylated first because it lies consistently closer to Pgamma. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation.
CONCLUSIONS AND SIGNIFICANCE - We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.
We report that knocking down the expression of inositol polyphosphate 4-phosphatase type II (INPP4B) in human epithelial cells, like knockdown of PTEN, resulted in enhanced Akt activation and anchorage-independent growth and enhanced overall motility. In xenograft experiments, overexpression of INPP4B resulted in reduced tumor growth. INPP4B preferentially hydrolyzes phosphatidylinositol-3,4-bisphosphate (PI(3,4)P(2)) with no effect on phosphatidylinositol-3.4.5-triphosphate (PI(3,4,5)P(3)), suggesting that PI(3,4)P(2) and PI(3,4,5)P(3) may cooperate in Akt activation and cell transformation. Dual knockdown of INPP4B and PTEN resulted in cellular senescence. Finally, we found loss of heterozygosity (LOH) at the INPP4B locus in a majority of basal-like breast cancers, as well as in a significant fraction of ovarian cancers, which correlated with lower overall patient survival, suggesting that INPP4B is a tumor suppressor.
The cardiac ryanodine receptor (RyR) controls Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling. Three phosphorylation sites have been identified: Serine-(S)2808, S2814 and recently S2030. We measured phosphorylation with at least two different antibodies per site and demonstrate that for S2808 results were highly antibody-dependent and two out of three S2808 antibodies did not accurately report phosphorylation level. The RyR was substantially phosphorylated in quiescent rat cardiomyocytes at S2808 and less so at S2814, but appeared to be unphosphorylated at S2030. Basal phosphorylation at S2808/S2814 was maintained by a Ca2+ dependent kinase other than Ca2+/Calmodulin-dependent kinase (CaMKII). During stimulation with Isoproterenol S2808 was phosphorylated by protein kinase A (PKA) and S2814 was phosphorylated by CaMKII. Phosphatase 1 appears to be the main phosphatase dephosphorylating S2808/S2814, but phosphatase 2a may also dephosphorylate S2814. RyR phosphorylation is complex, but important in understanding RyR functional modulation.
Phosphoinositide 3-kinase (PI3K) activation and synthesis of phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) lipids mediate growth factor signaling that leads to cell proliferation, migration, and survival. PI3K-dependent activation of Akt is critical for myoblast differentiation induced by serum withdrawal, suggesting that in these cells PI3K signaling is activated in an unconventional manner. Here we investigate the mechanisms by which PI3K signaling and Akt are regulated during myogenesis. We report that PI-3,4-P2 and PI-3,4,5-P3 accumulated in the plasma membranes of serum-starved 3T3-L6 myoblasts due to de novo synthesis and increased lipid stability. Surprisingly, only newly synthesized lipids were capable of activating Akt. Knockdown of the lipid phosphatase PTEN moderately increased PI3K lipids but significantly increased Akt phosphorylation and promoted myoblast differentiation. Knockdown of the lipid phosphatase Ship2, on the other hand, dramatically increased the steady-state levels of PI-3,4,5-P3 but did not affect Akt phosphorylation and increased apoptotic cell death. Together, these results reveal the existence of two distinct pools of PI3K lipids in differentiating 3T3-L6 myoblasts: a pool of nascent lipids that is mainly dephosphorylated by PTEN and is capable of activating Akt and promoting myoblast differentiation and a stable pool that is dephosphorylated by Ship2 and is unable to activate Akt.