scholarly journals A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

2020 ◽  
Vol 295 (34) ◽  
pp. 12262-12278
Author(s):  
Surya P. Manandhar ◽  
Ikha M. Siddiqah ◽  
Stephanie M. Cocca ◽  
Editte Gharakhanian
Keyword(s):  
Membrane Fusion ◽  
Phosphorylation Site ◽  
Casein Kinase ◽  
Membrane Dynamics ◽  
Vacuolar Membrane ◽  
Site Directed Mutagenesis ◽  
Casein Kinase I ◽  
C Terminus ◽  
Kinase Cascade

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3–Env7 kinase cascade.

scholarly journals Vps41 Phosphorylation and the Rab Ypt7 Control the Targeting of the HOPS Complex to Endosome–Vacuole Fusion Sites

2009 ◽  
Vol 20 (7) ◽  
pp. 1937-1948 ◽  
Author(s):  
Margarita Cabrera ◽  
Clemens W. Ostrowicz ◽  
Muriel Mari ◽  
Tracy J. LaGrassa ◽  
Fulvio Reggiori ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Hot Spot ◽  
Phosphorylation Site ◽  
Casein Kinase ◽  
Vacuole Fusion ◽  
Fusion Defect ◽  
Fusion Site ◽  
Vacuole Biogenesis ◽  
Hops Complex

Membrane fusion depends on multisubunit tethering factors such as the vacuolar HOPS complex. We previously showed that the vacuolar casein kinase Yck3 regulates vacuole biogenesis via phosphorylation of the HOPS subunit Vps41. Here, we link the identified Vps41 phosphorylation site to HOPS function at the endosome–vacuole fusion site. The nonphosphorylated Vps41 mutant (Vps41 S-A) accumulates together with other HOPS subunits on punctate structures proximal to the vacuole that expand in a class E mutant background and that correspond to in vivo fusion sites. Ultrastructural analysis of this mutant confirmed the presence of tubular endosomal structures close to the vacuole. In contrast, Vps41 with a phosphomimetic mutation (Vps41 S-D) is mislocalized and leads to multilobed vacuoles, indicative of a fusion defect. These two phenotypes can be rescued by overproduction of the vacuolar Rab Ypt7, revealing that both Ypt7 and Yck3-mediated phosphorylation modulate the Vps41 localization to the endosome–vacuole junction. Our data suggest that Vps41 phosphorylation fine-tunes the organization of vacuole fusion sites and provide evidence for a fusion “hot spot” on the vacuole limiting membrane.

scholarly journals Phosphorylation of the MAPKKK Regulator Ste50p in Saccharomyces cerevisiae: a Casein Kinase I Phosphorylation Site Is Required for Proper Mating Function

2003 ◽  
Vol 2 (5) ◽  
pp. 949-961 ◽  
Author(s):  
Cunle Wu ◽  
Mathieu Arcand ◽  
Gregor Jansen ◽  
Mei Zhong ◽  
Tatiana Iouk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Casein Kinase ◽  
Casein Kinase I ◽  
Aspartic Acid Residue ◽  
Glycerol Synthesis ◽  
Mating Function

ABSTRACT The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.

scholarly journals Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole

2012 ◽  
Vol 33 (3) ◽  
pp. 526-542 ◽  
Author(s):  
Surya P. Manandhar ◽  
Florante Ricarte ◽  
Stephanie M. Cocca ◽  
Editte Gharakhanian
Keyword(s):  
Protein Kinase ◽  
Membrane Fusion ◽  
Phylogenetic Analyses ◽  
Vacuolar Membrane ◽  
Site Directed Mutagenesis ◽  
Organelle Biogenesis ◽  
Gene Encoding ◽  
Dynamic Structures

ABSTRACTMembrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15.In vitrokinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome systemin vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established thatenv7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds.ENV7function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, andENV7is not a suppressor ofyck3Δ. Bayesian phylogenetic analyses strongly supportENV7as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.

scholarly journals Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

1999 ◽  
Vol 19 (1) ◽  
pp. 526-536 ◽  
Author(s):  
Hideko Kasahara ◽  
Seigo Izumo
Keyword(s):  
Mutational Analysis ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Homeobox Gene ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Site Directed Mutagenesis ◽  
Phosphopeptide Mapping

ABSTRACT Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.

scholarly journals Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2Cα

1996 ◽  
Vol 320 (3) ◽  
pp. 801-806 ◽  
Author(s):  
Anna E. MARLEY ◽  
Jane E. SULLIVAN ◽  
David CARLING ◽  
W. Mark ABBOTT ◽  
Graeme J. SMITH ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Protein A ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Paramecium Tetraurelia ◽  
C Terminus ◽  
Specific Inhibitors

The use of protein phosphatase inhibitors has been instrumental in defining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B. Identification of the role of PP2C in vivo has been hampered, in part, by the unavailability of specific inhibitors. In order to facilitate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further characterize this enzyme at the molecular level by site-directed mutagenesis and X-ray crystallography, we have expressed active recombinant human PP2Cα (rPP2Cα) in Escherichia coli. Biochemical characterization of rPP2Cα showed that it could hydrolyse p-nitrophenyl phosphate (pNPP) although, in contrast with native PP2C, this was not stimulated by Mg2+. As with native PP2C, okadaic acid failed to inhibit rPP2Cα, whereas 50 mM NaF dramatically inhibited its activity. An alignment of the amino acid sequence of AMP-activated protein kinase (AMPK) with those of other serine/threonine protein kinases around the regulatory phosphorylation site (subdomains VII–VIII) revealed a high degree of conservation. Phosphopeptides derived from this region of AMPK and containing the almost invariant threonine (Thr172 in AMPK) were found to be good substrates for rPP2Cα. We also showed that rPP2Cα can inactivate AMPK, but only in the presence of Mg2+. To define the regions of PP2Cα important for catalytic activity, we expressed a number of truncated proteins based on the sequence and proposed domain structure of the PP2Cα homologue from Paramecium tetraurelia. Deletion of 75 residues (9 kDa) from the C-terminus appeared to have little effect on the catalytic activity using pNPP, phosphopeptides or AMPK as substrates. This suggests that the residues important in catalysis lie elsewhere in the protein. A further deletion of the C-terminus led to a completely inactive and very poorly soluble protein.

scholarly journals T202. THE EFFECT OF ANTIPSYCHOTIC DRUGS ON MEMBRANE FUSION: AN IN VITRO STUDY

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S308-S309
Author(s):  
Vladimir Adrien ◽  
Hugo Fumat ◽  
Cédric Tessier ◽  
Philippe Nuss ◽  
David Tareste
Keyword(s):  
Membrane Fusion ◽  
Lipid Composition ◽  
Synaptic Vesicles ◽  
Significant Proportion ◽  
Membrane Dynamics ◽  
Snare Proteins ◽  
Synaptic Membranes ◽  
Synaptic Plasma Membrane

Abstract Background Common clinical use of antipsychotics (AP) drugs shows that their therapeutic mode of action still needs further clarification although it is admitted that the Dopamine receptor D2 (D2R) antagonism plays a significant role. For instance, clozapine (CLOZ) - which is known to be the most effective AP in treating schizophrenic symptoms - has strikingly the lowest D2R antagonism. Non direct receptor-related effects might thus be involved in the activity of AP at the synapse level. AP, as well as neurotransmitters, are mostly lipophilic and insert within membranes. This characteristic is of interest as a significant proportion of schizophrenic patients has specific and abnormal membrane lipid composition. This possible proxy of the disease biotype can participate in the disease’s physiopathology but also be critical for the effect of AP drugs. We hypothesize that AP insertion into lipid membranes also contribute to their therapeutic effect. AP-induced modifications of synaptic membranes biophysics are likely to influence neurotransmission. In this study, we focus on the effect of AP on membrane fusion, a crucial step for the exocytosis of neurotransmitters. Methods Liposomes modelling synaptic vesicles were reconstituted in saline buffer. Two standard ternary and quaternary lipid mixtures have been studied: phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine (PC:PE:PS) [65:25:10] and the synaptic-like PC:PE:PS:sphingomyelin:cholesterol (PC:PE:PS:SM:CHOL) [25:25:10:10:30]. Some liposomes were protein-free and others were functionalized with Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) proteins, which trigger in vivo the fusion of synaptic vesicles with the pre-synaptic plasma membrane. The liposome size was checked by Dynamic Light Scattering. Insertion of AP within the membrane was checked by second derivative spectroscopy. Fusion was measured by Fluorescence Resonance Energy Transfer in the absence or presence of CLOZ or chlorpromazine (CPZ) at various lipid:AP ratios (10:1 to 100000:1). Protein-free liposomes were fused with Polyethylene glycol (PEG) and SNARE liposomes through the action of cognate SNARE proteins residing in their membrane. Results Liposomes of the same lipid composition were of the same size, with no effect of the addition of AP drugs at various concentrations. Molar partition coefficient of AP drugs within the membrane of protein-free liposomes was approximately 70–85%. CPZ or CLOZ inhibited the fusion of PC:PE:PS liposomes by about 20–40%. When liposomes were synaptic-like (PC:PE:PS:SM:CHOL), the inhibition of fusion by AP drugs reached 50%. CLOZ also inhibited SNARE-mediated fusion of PC:PE:PS liposomes by about 30%. This effect on SNARE-mediated fusion was not observed with CPZ. Discussion Altogether, these results, despite preliminary, could help to understand partially a non direct receptor-related effect of antipsychotics. Indeed, these drugs also seem to modify membrane dynamics at the synapse level. This seems to be particularly the case of CLOZ on SNARE-mediated fusion and could explain its specific therapeutic efficiency.

scholarly journals Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection

2001 ◽  
Vol 75 (24) ◽  
pp. 12188-12197 ◽  
Author(s):  
Tara L. Cartee ◽  
Gail W. Wertz
Keyword(s):  
Protein Function ◽  
Consensus Sequence ◽  
Casein Kinase ◽  
Rnase A ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Casein Kinase I ◽  
N Protein ◽  
Syncytial Virus

ABSTRACT The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in Escherichia coli could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1–N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.

scholarly journals The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex

2005 ◽  
Vol 168 (3) ◽  
pp. 401-414 ◽  
Author(s):  
Tracy J. LaGrassa ◽  
Christian Ungermann
Keyword(s):  
Cellular Membrane ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Casein Kinase I ◽  
Functional Protein ◽  
Hypertonic Stress ◽  
Functional Studies ◽  
Vacuole Fusion

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.

scholarly journals Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation

1994 ◽  
Vol 14 (3) ◽  
pp. 1594-1602
Author(s):  
A J Rossomando ◽  
P Dent ◽  
T W Sturgill ◽  
D R Marshak
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Dual Specificity ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade ◽  
Protein Kinase Kinase

Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.

Mechanisms of Apoptosis Induction by BH3 Inhibitor ABT-737 in AML.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 244-244
Author(s):  
Michael Andreeff ◽  
Rooha Contractor ◽  
Peter P. Ruvolo ◽  
Xingming Deng ◽  
Ismael Samudio ◽  
...  
Keyword(s):  
Cell Lines ◽  
Phosphorylation Site ◽  
Leukemia Cell ◽  
Mek Inhibitor ◽  
Chemotherapeutic Agents ◽  
Site Directed Mutagenesis ◽  
Apoptotic Pathway ◽  
Induced Apoptosis ◽  
In Cells

Abstract Bcl2 family proteins are key regulators of apoptosis. Aberrations in Bcl2 levels are known to promote tumorigenesis and chemoresistance. Thus, strategies to target Bcl2 will likely provide effective therapies for malignancies such as acute myeloid leukemia (AML). In this report, we investigate mechanisms of action of the novel small molecule Bcl2 inhibitor ABT-737 in AML. ABT-737 effectively killed AML patient blast cells and colony-forming cell lines at nanomolar concentrations with no effect on normal hematopoietic cells. Notably, CD34+38−123+ AML stem cells are highly sensitive to the compound. ABT-737-induced apoptosis is initiated by disruption of Bcl2:Bax dimers and activation of the intrinsic apoptotic pathway. ABT-737 works synergistically with chemotherapeutic agents such as ara-C and doxorubicin. To investigate the role of Bcl-2 phosphorylation in the sensitivity to BH3 inhibitor, we used IL-3 dependent NSF.N1/H7 mouse myeloid cells modified by site-directed mutagenesis to produce various Bcl-2 phospho-mutants. NSF.N1/H7 cells stably transfected with phosphomimetic T69E/S70E/S87E (EEE) Bcl-2 mutants were resistant to ABT-737 (IC50>500 nM) as compared to cells expressing wt-Bcl-2 or the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutants (IC50s of 50 and 25 nM). Consistent with a mechanism whereby increased Bcl2 phosphorylation impedes ABT-737 suppression of Bcl2 dimerization with Bax, ABT-737 potently blocked Bcl2:Bax association in cells expressing exogenous WT Bcl2 and AAA mutant Bcl2 but not in cells expressing exogenous phosphomimetic EEE mutant Bcl2. Since the S70E phosphorylation site of Bcl-2 is a known ERK substrate, we examined combined effects of ABT-737 and MEK inhibitor PD98059 in OCI-AML3 cells resistant to ABT-737 alone. The combined activity of PD98059 and ABT-737, evaluated by isobologram analysis, revealed a striking synergistic interaction between the MEK and BH3 inhibitors, with combination indices (CI) of 0.08±0.003. OCI-AML3 cells exhibit the highest expression of Mcl-1 among the acute leukemia cell lines tested. We propose that loss of Mcl-1 expression as a result of suppression of ERK may also be involved in the ability of PD98059 to enhance ABT-737-induced apoptosis. siRNA to Mcl-1 strikingly sensitized OCI-AML3 cells to ABT-induced apoptosis (14% apoptosis in parental cells at 2.5μM ABT-737, 64% apoptosis in siRNA-transfected cells at 10-fold lower concentration of 0.25μM). We have further demonstrated that ABT-737 reduced leukemia burden and significantly (p=0.0018) prolonged survival of mice in an in vivo mouse model. These findings suggest that: 1) ABT-737 reduces apoptosis through disruption of Bcl2:Bax heterodimers; 2) its activity is limited by Bcl2 phosphorylation and Mcl-1 overexpression; 3) combination with MEK inhibition results in inhibition of Bcl2 phosphorylation, downregulation of Mcl-1 and dramatic enhancement of ABT-737-induced apoptosis in AML.

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