Ahnak Protein Activates Protein Kinase C (PKC) through Dissociation of the PKC-Protein Phosphatase 2A Complex

Lee, In Hye; Lim, Ho-Jin; Yoon, Sang-Hoon; Seong, Je Kyung; Bae, Duk Soo; Rhee, Sue Goo; Bae, Yun Soo

doi:10.1074/jbc.m706878200

Cited by 56 publications

(51 citation statements)

References 46 publications

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“…Because PKCa/b activity can be modulated by its phosphorylation state, as well as by its cell localization (50) and association with specific adapters (51), we decided to explore whether Fyn could modulate the interaction between PP2A and PKCa/b, as well as the association of PKCa/b with proteins that could increase its activity. In this regard, the role of the adapter protein AHNAK (desmoyokin) in the increase in PKC activity as a result of its dissociation from PP2A was described (52). We decided to explore whether LPS-induced triggering of TLR4 in BMMCs could lead to an association between AHNAK and PKCa/b and whether this could be altered in Fyn-deficient cells.…”

Section: Resultsmentioning

confidence: 99%

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

Martín-Ávila

Medina-Tamayo

Ibarra-Sánchez

et al. 2016

The Journal of Immunology

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Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow–derived mast cells from wild-type and Fyn-deficient mice. Fyn−/− cells showed higher LPS-induced secretion of preformed and de novo–synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn−/− cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn−/− cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/β, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation–associated protein (AHNAK, desmoyokin). LPS-induced PKCα/β activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn−/− MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/β necessary for the secretion of TNF by VAMP3+ carriers.

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Section: Resultsmentioning

confidence: 99%

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

Martín-Ávila

Medina-Tamayo

Ibarra-Sánchez

et al. 2016

The Journal of Immunology

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“…The precise function of PDAP1 is unclear, but it has been reported to be highly up-regulated in the secretome of neoplastic gastric epithelial cells (58), a context in which SMYD2 activity was recently characterized (26). Finally, AHNAK and AHNAK2, which are uniquely and extensively directly mono-methylated by SMYD2, appear to possess diverse functionality (reviewed in (59)) with roles ranging from cell adhesion (60), cell signaling (61,62), and tumor cell migration and invasion (63). Whether the extensive mono-methylation of the AHNAK and AHNAK2 CRUs by SMYD2, as well as the individual sites we identified in other SMYD2 substrates, regulates the function of these and other proteins remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

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The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9). The majority of PKMT substrates have been identified based on biochemical methylation assays using recombinant enzyme and substrate, followed by cell-based assays typically requiring overexpression of enzyme and/or substrate to maximize signal detection (10 -13). However, it is difficult to discern whether these substrates are bona fide physiological substrates of endogenous PKMT activity. With the development of PKMT-targeted drugs emerging as a key area of drug discovery (14 -18), unbiased and quantitative methods enabling the comprehensive identification of histone and non-histone substrates in cells are critical to clarifying the cell-relevant substrates of these enzymes and to more accurately understand the functions of non-histone methylation.Progressing from targeted biochemical...

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“…Classically, PKCs activate MEK through a signaling cascade involving Raf-1, but in our study, we did not find differences in Raf-1 activation between STC1 þ / þ and STC1À/À MEFs, so if PKC is involved, it appears to be independent of Raf-1 function. PKC is also known to interact with protein phosphatase 2A (Lee et al, 2008), and it is possible that STC1 cooperates with this enzyme to inhibit the phosphorylation and activation of MEK.…”

Section: Regulation Of Oxidative Stress Response By Stc1mentioning

confidence: 99%

Stanniocalcin-1 acts in a negative feedback loop in the prosurvival ERK1/2 signaling pathway during oxidative stress

2009

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Mammalian Stanniocalcin-1 (STC1) is a glycoprotein that has been implicated in various biological processes including angiogenesis. Aberrant STC1 expression has been reported in breast, ovarian and prostate cancers, but the significance of this is not well understood. Here, we report that oxidative stress caused a 40-fold increase in STC1 levels in mouse embryo fibroblasts (MEFs). STC1À/À MEFs were resistant to growth inhibition and cell death induced by H 2 O 2 or by 20% O 2 (which is hyperoxic for most mammalian cells); this is the first phenotype reported for STC1-null cells. STC1À/À cells had higher levels of activated MEK and ERK1/2 than their wild-type (WT) counterparts, and these levels were all reduced by stable expression of exogenous STC1 in STC1À/À cells. Furthermore, pharmacological inhibition by PD98059 or UO126 of MEK and therefore of ERK1/2 activation restored sensitivity of STC1À/À cells to oxidative stress. We also found that H 2 O 2 -induced STC1 expression in WT cells was abolished by inhibition of ERK1/2 activation. Thus, the ERK1/2 signaling pathway upregulates STC1 expression, which in turn downregulates the level of activated MEK and consequently ERK1/2 in a novel negative feedback loop. Therefore, STC1 expression downregulates prosurvival ERK1/2 signaling and reduces survival under conditions of oxidative stress.

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Ahnak Protein Activates Protein Kinase C (PKC) through Dissociation of the PKC-Protein Phosphatase 2A Complex

Cited by 56 publications

References 46 publications

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Stanniocalcin-1 acts in a negative feedback loop in the prosurvival ERK1/2 signaling pathway during oxidative stress

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