Role of SH2 Domain in JAK2-V617F Mediated Transformation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3608-3608 ◽  
Author(s):  
Sivahari P. Gorantla ◽  
Tobias Dechow ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Point Mutation ◽  
Jak2 V617f ◽  
Cellular Transformation ◽  
Sh2 Domain ◽  
Cytokine Receptor ◽  
Wild Type ◽  
Therapeutic Implications ◽  
Proliferation Assays ◽  
In Cells

Abstract A point mutation in JAK2 (V617F) has been described recently in patients with myeloproliferative diseases like polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (IMF). This V617F point mutation in JAK2 has been shown to activate several downstream pathways including STAT5 and ERK. This mutation also renders haematopoietic progenitors cytokine-independent. The role of the V617F mutation in oncogenesis is not fully understood. In this study we aim to dissect the role of the SH2 domain in JAK2-V617F mediated transformation. Stable Ba/F3 cell lines expressing JAK2-wild type (wt), JAK2-V617F, JAK2-R439K (SH2 domain mutation) and JAK2-V617F/R439K mutants were generated. Cell proliferation assays showed that JAK2-V617F transforms Ba/F3 cells and renders them IL3 independent, while wild type JAK2 and JAK2-R439K could not. Surprisingly, JAK2-V617F/R439K was not able to induce a transformed phenotype in Ba/F3 cells. Imunoblotting revealed strong activation of JAK2, STAT5 and ERK in cells expressing JAK2-V617F, whereas no such activation could be found in JAK2-wt, JAK2-R439K and in JAK2-V617F/R439K expressing cells. Thus the SH2 domain in JAK2-V617F seems to play a crucial role in the transformation of Ba/F3 cells containing a heterodimeric (IL-3) cytokine receptor. It has been demonstrated that JAK2-V617F induces cellular transformation more efficiently in cells expressing a homodimeric cytokine receptor such as the erythropoetin receptor. We therefore established Ba/F3 cells overexpressing EpoR together with JAK2-wt, JAK2-V617F, JAK2-R439K and JAK2-V617F/R439K. In contrast to parental Ba/F3 cells, EpoR expressing Ba/F3 cells could be transformed by both JAK2-V617F as well as JAK2-V617F/R439K. Both the single and double mutant Ba/F3 cells showed strong activation of STAT5 and ERK. This suggests that an intact SH2 domain is not required for homodimeric cytokine receptor expressing cells. These results show that transformation by JAK2-V617F requires an intact SH2 domain only in cells expressing a heterodimeric cytokine receptor. In contrast, cells containing a homodimeric cytokine receptor are able to induce transformation in the presence of JAK2-V617F with an additional SH2 mutation. Further progress in understanding the role of the SH2 domain in JAK2-V617F mediated transformation may help in delineating downstream signalling with therapeutic implications.

Adaptor Protein LNK Binds to and Is Phosphorylated by JAK3 and May Serve as a Scaffold for JAK3 Autophosphorylation In the Absence of An Appropriate Cytokine Receptor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2785-2785 ◽  
Author(s):  
Maya koren-Michowitz ◽  
Sigal Gery ◽  
Daniel Nowak ◽  
Arnon Nagler ◽  
Aiko Matsubara ◽  
...  
Keyword(s):  
T Cell ◽  
Point Mutation ◽  
Cell Lymphoma ◽  
Adaptor Protein ◽  
Jak2 V617f ◽  
Sh2 Domain ◽  
Cytokine Receptor ◽  
The Other ◽  
Ph Domain ◽  
Hematopoietic Stem

Abstract Abstract 2785 The adaptor protein LNK has an important role during the development and maturation of cells in the hematopoietic system. LNK KO mice have an expansion of hematopoietic stem cells (HSCS) and an increase in circulating neutrophils, platelets, as well as immature B cells. On the other hand, overexpression of WT LNK under the control of a lymphocyte-specific expression vector results in an impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. Recently, LNK PH domain mutations were described in myeloproliferative neoplasms (MPN). The non-receptor tyrosine kinase JAK3 is expressed mainly in hematopoietic cells and together with JAK1, transmits signals from cytokine receptors of the IL-2 family. While JAK3 loss of function causes severe combined immune deficiency (SCID), JAK3 activating mutations were described in AML, particularly in acute megakaryocytic leukemia (AMKL). The importance of both LNK and JAK3 in lymphopoiesis, coupled with the finding that LNK binds to another JAK family member (JAK2), has led us to study whether LNK and JAK3 interact. LNK and JAK3 co- immunoprecipitated (co-IP) in the following leukemic cell lines- HEL, NALM6 (containing both LNK and WT JAK3), CMK (containing both LNK and an activating JAK3 mutant) and not in K562 (no JAK3). WT-JAK3 and WT LNK also co-IP when overexpressed in 293T cells, and this binding was associated with phosphorylation of LNK. To determine which domain of LNK is responsible for JAK3 binding, WT JAK3 was overexpressed in 293T cells with the following LNK mutants: 1. point mutation in the SH2 domain (R392E); 2. Deletion of the SH2 domain (del SH2); 3. Deletion of the PH and SH2 (del SH2/PH) domains; 4. Point mutation in the PH domain, recently found in a patient with a JAK2 V617F negative MPN (E208Q); 5. LNK SNP (rs3184504) described in GWAS to associate with an increased risk of autoimmune disease and which also causes an amino acid change in the LNK PH domain (W262R). LNK binding was retained in the E208Q and W262R mutants, markedly reduced in the R392E and del SH2 mutants and completely abolished with the del SH2/PH mutant, suggesting that LNK SH2 domain is important for the binding of LNK and WT-JAK3, although some binding may occur even in its absence. The effect of the LNK mutants R392E, E208Q and W262R on WT-JAK3 and LNK phosphorylation was also studied in 293T cells. Notably, WT-JAK3 was phosphorylated in the presence of WT-LNK and the E208Q and W262R mutants and not in the presence of the R392E mutant or an empty vector; also, the WT-LNK, LNK E208Q and W262R were phophorylated and the LNK R392E was not. These results suggest that WT-JAK3 phosphorylates LNK and that the LNK SH2 domain is important for this to occur. Interestingly, we found that the LNK PH domain mutant (E208Q) was much more phosphorylated than the WT- LNK when co-expressed with WT-JAK3. A similar result was obtained when LNK E208Q was co-expressed in 293T cells with vectors expressing an activating JAK3 mutant- A572V and the JAK2 V617F mutant but not when it was co-expressed with a BCR-ABL vector. This, again, supports the fact that direct binding underlines LNK phosphorylation by JAK3, as was previously shown for JAK2, while the BCR-ABL kinase does not bind LNK. The LNK PH domain mutation E208Q may cause a conformational change in LNK, allowing better access to tyrosine residues, or a change in LNK localization. The finding in 293T cells that WT-JAK3 is phophorylated only in the presence of LNK that retains a PH domain is intriguing. The other LNK family members- SH2B1 and APS were previously shown to homo- and heterodimerize. LNK contains a dimerization domain and we speculate that LNK homodimerization may serve as a scaffold for WT-JAK3 in the absence of a cytokine receptor, enabling JAK3 autophosphorylation. Since LNK PH domain mutations were recently described in MPN and may involve activation of the JAK2-STAT pathway we hypothesized that similar mutations might be found in lymphoma cases where JAK3 is reported to be activated. We, therefore, sequenced LNK PH domain in 147 lymphoma samples including- mantle cell lymphoma, peripheral T cell lymphoma (PTCL) and adult T cell leukemia/lymphoma cases but could not detect any mutations. In summary- we found that JAK3 binds and phosphorylates both WT-LNK and to a greater extent, the LNK PH domain mutant E208Q and in the absence of a cytokine receptor can lead to JAK3 phosphorylation. Taken together, LNK may likely control JAK3 activity. Disclosures: No relevant conflicts of interest to declare.

Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 175-175
Author(s):  
Sivahari P Gorantla ◽  
Tobias Dechow ◽  
Rebekka Grundler ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Jak2 V617f ◽  
Sh2 Domain ◽  
Cytokine Receptor ◽  
Cytokine Receptors ◽  
Type I ◽  
Constitutive Activation ◽  
Downstream Signaling

Abstract The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.

The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain

1993 ◽  
Vol 105 (2) ◽  
pp. 519-528
Author(s):  
F. Boschelli ◽  
S.M. Uptain ◽  
J.J. Lightbody
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Sh2 Domain ◽  
Wild Type ◽  
Type V ◽  
Cdc28 Kinase ◽  
In Cells

The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints. Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine. Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed. The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain. Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity. These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2172-2180 ◽  
Author(s):  
Kotaro Suzuki ◽  
Hiroshi Nakajima ◽  
Norihiko Watanabe ◽  
Shin-ichiro Kagami ◽  
Akira Suto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cytokine Receptor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Wild Type ◽  
Peritoneal Mast Cells ◽  
The Common

Abstract The regulatory roles of the common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γc−) and Jak3-deficient (Jak3−) mice. Although the mast cells in γc− and Jak3− mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc− and Jak3− mice as compared with that in wild-type mice. Among γc-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow–derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γc− and Jak3−mice. In addition, IL-4Rα, γc, and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γc− and Jak3− mice. These results indicate that γc- and Jak3-dependent signaling is essential for IL-4– and IL-9–induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γc- and Jak3-dependent manner.

scholarly journals Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia.

1994 ◽  
Vol 125 (5) ◽  
pp. 1057-1065 ◽  
Author(s):  
S C Dahl ◽  
R W Geib ◽  
M T Fox ◽  
M Edidin ◽  
D Branton
Keyword(s):  
Membrane Structure ◽  
Membrane Skeleton ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Erythroleukemia Cells ◽  
Alpha Spectrin ◽  
Erythroleukemia Cell ◽  
The Stability ◽  
In Cells

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

scholarly journals Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.

1989 ◽  
Vol 9 (2) ◽  
pp. 629-638 ◽  
Author(s):  
A B Reynolds ◽  
D J Roesel ◽  
S B Kanner ◽  
J T Parsons
Keyword(s):  
Tyrosine Phosphorylation ◽  
Intracellular Localization ◽  
Cellular Transformation ◽  
Cellular Protein ◽  
Morphological Transformation ◽  
Specific Antibodies ◽  
Phosphorylated Proteins ◽  
Src Gene ◽  
In Cells

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

Allele-specific wild-type blocker quantitative PCR for highly sensitive detection of rare JAK2 p.V617F point mutation in primary myelofibrosis as an appropriate tool for the monitoring of molecular remission following therapy

2010 ◽  
Vol 63 (4) ◽  
pp. 370-372 ◽  
Author(s):  
Udo Siebolts ◽  
Thoralf Lange ◽  
Dietger Niederwieser ◽  
Claudia Wickenhauser
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Point Mutation ◽  
Cell Transplantation ◽  
Jak2 V617f ◽  
Clinical Relapse ◽  
Wild Type ◽  
Bone Marrow Biopsies ◽  
Allele Specific

Screening of JAK2 V617F point mutation becomes more and more important in monitoring of JAK2 positive MPN following stem cell transplantation. In an attempt to achieve the required high sensitivity (1:105), specifity and robustness we created an approach applicable on bone marrow biopsies where we adapted the principle of wild-type blocker PCR with allele-specific Q-PCR. The significance of the assay was demonstrated on a retrospective series of sequential bone marrow biopsies as diagnosis of molecular relapse now preceded the diagnosis of clinical relapse by far. This method offers the urgently needed tool for a systematic molecular analysis of sequential biopsies in the course of stem cell transplantation to develop guidelines for the management of these patients.

scholarly journals The Role of Putative Phosphatidylserine-Interactive Residues of Tissue Factor on Its Coagulant Activity at the Cell Surface

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 213-213
Author(s):  
Shabbir Ansari ◽  
Usha R. Pendurthi ◽  
L. Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Specific Activity ◽  
Direct Interaction ◽  
Lipid Binding ◽  
Wild Type ◽  
Binding Region ◽  
Coagulant Activity ◽  
In Cells

Abstract Tissue factor (TF) is the cellular cofactor for the serine protease coagulation factor VIIa (FVIIa). The TF-FVIIa complex formed on the cell surface initiates the coagulation cascade. It is believed that most of the TF molecules on the cell surface of a resting cell exist in an encrypted state with very little procoagulant activity. Encrypted TF must undergo decryption to become fully active. The exact mechanisms by which TF activity on the cell surface is regulated are unknown. Exposure of phosphatidylserine (PS) to the outer leaflet of the cell membrane is thought to play a critical role in TF decryption. Recent studies of molecular dynamics simulation of TF ectodomain in solution and on the surface of anionic phospholipids suggested a direct interaction of PS headgroups with specific residues in TF. At present, the role of the putative lipid interactive residues of TF in TF decryption is unknown. In the present study, we investigated the potential role of TF direct interaction with the cell surface lipids on basal TF activity as well as enhanced TF activity following the decryption using different TF mutants. Plasmids or adenoviral constructs encoding wild-type or mutant TF (mutations in the putative lipid binding region) were used to transduce TF expression in CHO-K1 or monocytic THP-1 cells, respectively. TF protein expression level at the cell surface and FVIIa binding to the cell surface TF were evaluated by radioligand binding studies using 125I-labeled TF mAb or FVIIa, respectively. TF-FVIIa coagulant activity on the cell surface was determined in FX activation assay. Data of these studies showed that all TF mutants were capable of interacting with FVIIa with no apparent defect. Out of the 9 selected TF mutants, five of them -TFS160A, TFS161A, TFS162A, TFK165A, and TFD180A-exhibited a similar or slightly higher TF coagulant activity to that of the wild-type TF. The specific activity of three mutants, TFK159A, TFS163A and TFK166A, was reduced substantially to a range of 40% - 70% of that of wild-type TF. Mutation of the glycine residue at the position 164 markedly abrogated the TF coagulant activity, resulting in ~90% loss of TF specific activity. Mutation of all nine lipid binding residues together (DLBR) did not further decrease the specific activity of TF anymore than that of mutation of G164 alone. Comparison of the present data with the published data on these mutants revealed that some of the TF residues that are critical for regulating TF activity on liposomes are not crucial for TF activity on the cell surface. To address whether the decreased FXa generation seen with the select TF variants is caused by changes in TF-membrane interaction or by the substrate interaction with TF/FVIIa complex, we performed Michaelis-Menten kinetics of FX activation for two of TF mutants (TFS163A and TFG164A). Results of this study suggested that there were no significant differences in Km values between wild-type TF and TF mutants (wild-type TF, 51 ± 14.6 nM; TFS163A, 68 ± 19.5 nM; TFG164A, 39 ± 18.4 nM, n=4). Interestingly, mutation of the selective residues in the lipid binding region failed to abrogate the PS-dependent TF decryption. The fold-increase in TF activity in cells expressing wild-type TF or TF variants was similar following cell activation with either HgCl2 or calcium ionomycin treatment. Annexin V markedly diminished the increased TF-FVIIa activation of FX in cells expressing wild-type TF as well as cells expressing the TF mutant (DLBR mutant). Overall, our data suggest that the regulation of TF activity at the cell surface milieu may be different from that of PC/PS vesicles and TF region other than earlier identified LBR may be responsible for enhancing TF activity following the PS exposure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of the Cold-Box Region in the 5′ Untranslated Region of the cspA mRNA in Its Transient Expression at Low Temperature in Escherichia coli

1998 ◽  
Vol 180 (1) ◽  
pp. 90-95 ◽  
Author(s):  
Li Fang ◽  
Yan Hou ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Untranslated Region ◽  
Cold Shock ◽  
Wild Type ◽  
Mrna Stabilization ◽  
High Level ◽  
Cold Box ◽  
In Cells

ABSTRACT Upon temperature downshift, a group of proteins called cold shock proteins, such as CspA, CspB, and CsdA, are transiently induced inEscherichia coli. However, when the 5′ untranslated region (5′ UTR) of cspA mRNA is overproduced at low temperature, the expression of cold shock genes is prolonged or derepressed. It has been proposed that this effect is due to highly conserved 11-base sequences designated the “cold box” existing in the 5′ UTRs ofcspA, cspB, and csdA. Here, we demonstrate that the overproduction of the 5′ UTR of not onlycspA but also cspB and csdA mRNAs causes derepression of all three genes at the same time. Conversely, when the cold-box region was deleted from the cspA 5′ UTR its derepression function was abolished. The amount of mRNA from the chromosomal cspA gene was much higher in cells overproducing the wild-type 5′ UTR by means of a plasmid than it was in cells overproducing the cold-box-deleted 5′ UTR. The stability of the chromosomal cspA mRNA in cells overproducing the wild-type 5′ UTR was almost identical to that in cells overproducing the cold-box-deleted 5′ UTR. Therefore, the derepression ofcspA caused by overproduction of 5′ UTR at the end of the acclimation phase occurs at the level of transcription but not by mRNA stabilization, indicating that the cold-box region plays a negative role in cspA transcription in cold shock-adapted cells. The role of the cold-box region was further confirmed with acspA mutant strain containing a cold-box-deletedcspA gene integrated into the chromosome, which showed a high level of constitutive production of CspA but not CspB during exponential growth at low temperature.

Abstract 174: The BH3-Only Protein BNIP3 Induces Mitochondrial Clearance via Multiple Pathways

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Eileen R Gonzalez ◽  
Babette Hammerling ◽  
Rita Hanna ◽  
Dieter A Kubli ◽  
Åsa B Gustafsson
Keyword(s):  
Cell Death ◽  
Ubiquitin Ligase ◽  
Critical Role ◽  
E3 Ubiquitin Ligase ◽  
Wild Type ◽  
Potent Inducer ◽  
Autophagy Pathway ◽  
Endosomal Pathway ◽  
In Cells

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.

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