Victor Bustos

Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation

Self‐renewal of Bcr‐Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine‐(S/T) unphosphorylated β‐catenin. Although β‐catenin can be tyrosine (Y)‐phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr‐Abl‐associated leukemogenesis is unknown. Here we show that Bcr‐Abl levels control the degree of β‐catenin protein stabilization by affecting its Y/S/T‐phospho content in CML cells. Bcr‐Abl physically interacts with β‐catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β‐catenin at Y86 and Y654 residues. This Y‐phospho β‐catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr‐Abl antagonist, impairs the β‐catenin/TCF‐related transcription causing a rapid cytosolic retention of Y‐unphosphorylated β‐catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr‐Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β‐catenin Y phosphorylation, Axin/GSK3β binding to β‐catenin and its subsequent S/T phosphorylation. Silencing of β‐catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr‐Abl+ CML cells, in synergism with Imatinib. These findings indicate the Bcr‐Abl triggered Y phosphorylation of β‐catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

A small-molecule enhancer of autophagy decreases levels of Aβ and APP-CTF via Atg5-dependent autophagy pathway

The hallmarks of Alzheimers disease are the aggregates of amyloid‐β (Aβ) peptide and tau protein. Autophagy is one major cellular pathway leading to the removal of aggregated proteins. We examined the possibility of inducing autophagy to reduce Aβ peptide and the amyloid precursor protein (APP)‐derived fragment APP‐CTF levels in cell lines and primary neuronal cultures. We found that induction of autophagy either by small‐molecule enhancers of rapa‐mycin (SMER)28, a small‐molecule enhancer of autophagy, or following starvation greatly decreased the levels of Aβ peptide (apparent EC50 of ~10 µM) and APP‐CTF (apparent EC50 of ~20 µM) in a γ‐secretase‐independent manner. Pharmacological inhibition of autophagy led to a significant accumulation of Aβ peptide and APP‐CTF and diminished the effect of SMER28. Three essential components of the autophagic pathway, autophagy‐related protein (Atg)5, Beclin1, and Ulk1, were shown to be involved in the degradation of Aβ and APP‐CTF, and Atg5 was necessary for the effect of SMER28. In addition, the autophagic marker light chain 3‐II cocompartmentalized with APP‐CTF. These results support the involvement of autophagy in the clearance of Aβ and APP‐CTF. We therefore propose that small molecule enhancers of autophagy, such as SMER28, may have therapeutic potential for the treatment of Alzheimers disease and other proteinopathies.—Tian, Y., Bustos, V., Flajolet, M., Greengard, P. A small‐molecule enhancer of autophagy decreases levels of Aβ and APP‐CTF via Atg5‐dependent autophagy pathway. FASEB J. 25, 1934‐1942 (2011). www.fasebj.org

A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of β-catenin, “priming” the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of β-catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n-3 from the target serine. However, the β-catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of β-catenin were assayed with purified rat liver CK1 or recombinant CK1α and CK1αL from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant β-catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of β-catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1.

Multiple phosphorylation of β-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38–64 of β-catenin are phosphorylated by CK1 on Ser-45 with low affinity (Km ≈1 mM), whereas intact β-catenin is phosphorylated at Ser-45 with very high affinity (Km ≈200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70–74 positions or a F74AA mutation in full-length β-catenin had no significant effect on CK1 phosphorylation efficiency. β-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131–181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in Km value. Implication of the first armadillo repeat in β-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of β-catenin from α-catenin, further improves CK1 phosphorylation efficiency, lowering the Km value to <50 nM, approximating the physiological concentration of β-catenin. In contrast, α-catenin, which interacts with the N-terminal region of β-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that β-catenin association with α-catenin and β-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

Reduction of Synaptojanin 1 Accelerates Aβ Clearance and Attenuates Cognitive Deterioration in an Alzheimer Mouse Model

Background: Recent studies have linked synaptojanin 1 (synj1) with Alzheimer disease (AD). Results: We report that synj1 reduction decreases amyloid plaque burden and attenuates cognitive deterioration in an AD mouse model. These effects are mediated through accelerating endosomal/lysosomal degradation of Aβ. Conclusion: Our data suggest a novel mechanism by which synj1 reduction promotes Aβ clearance. Significance: These studies implicate a therapeutic strategy for AD. Recent studies link synaptojanin 1 (synj1), the main phosphoinositol (4,5)-biphosphate phosphatase (PI(4,5)P2-degrading enzyme) in the brain and synapses, to Alzheimer disease. Here we report a novel mechanism by which synj1 reversely regulates cellular clearance of amyloid-β (Aβ). Genetic down-regulation of synj1 reduces both extracellular and intracellular Aβ levels in N2a cells stably expressing the Swedish mutant of amyloid precursor protein (APP). Moreover, synj1 haploinsufficiency in an Alzheimer disease transgenic mouse model expressing the Swedish mutant APP and the presenilin-1 mutant ΔE9 reduces amyloid plaque load, as well as Aβ40 and Aβ42 levels in hippocampus of 9-month-old animals. Reduced expression of synj1 attenuates cognitive deficits in these transgenic mice. However, reduction of synj1 does not affect levels of full-length APP and the C-terminal fragment, suggesting that Aβ generation by β- and γ-secretase cleavage is not affected. Instead, synj1 knockdown increases Aβ uptake and cellular degradation through accelerated delivery to lysosomes. These effects are partially dependent upon elevated PI(4,5)P2 with synj1 down-regulation. In summary, our data suggest a novel mechanism by which reduction of a PI(4,5)P2-degrading enzyme, synj1, improves amyloid-induced neuropathology and behavior deficits through accelerating cellular Aβ clearance.

APP intracellular domain–WAVE1 pathway reduces amyloid-β production

An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheimers disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aβ production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aβ. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aβ levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Aβ production.

Small-molecule inducers of Aβ-42 peptide production share a common mechanism of action

The pathways leading specifically to the toxic Aβ42 peptide production, a key event in Alzheimers disease (AD), are unknown. While searching for pathways that mediate pathological increases of Aβ42, we identified Aftin‐4, a new compound that selectively and potently increases Aβ42 compared to DMSO (N2a cells: 7‐fold; primary neurons: 4‐fold; brain lysates: 2‐fold) with an EC50 of 30 μM. These results were confirmed by ELISA and IP‐WB. Using affinity chromatography and mass spectrometry, we identified 3 proteins (VDAC1, prohibitin, and mitofilin) relevant to AD that interact with Aftin‐4, but not with a structurally similar but inactive molecule. Electron microscopy studies demonstrated that Aftin‐4 induces a reversible mitochondrial phenotype reminiscent of the one observed in AD brains. Sucrose gradient fractionation showed that Aftin‐4 perturbs the subcellular localization of γ‐secretase components and could, therefore, modify γ‐secretase specificity by locally altering its membrane environment. Remarkably, Aftin‐4 shares all these properties with two other “AD accelerator” compounds. In summary, treatment with three Aβ42 raising agents induced similar biochemical alterations that lead to comparable cellular phenotypes in vitro, suggesting a common mechanism of action involving three structural cellular targets.—Bettayeb, K., Oumata, N., Zhang, Y., Luo, W., Bustos, V., Galons, H., Greengard, P., Meijer, L., Flajolet, M. Small‐molecule inducers of Aβ‐42 peptide production share a common mechanism of action. FASEB J. 26, 5115–5123 (2012). www.fasebj.org

Phosphorylated Presenilin 1 decreases β-amyloid by facilitating autophagosome–lysosome fusion

Significance Autophagy is a catabolic process involving the formation of double-membrane–bound organelles called autophagosomes, which participate in the degradation of intracellular material through fusion with lysosomes. We have found a level of regulation of autophagosomal–lysosomal fusion where Presenilin 1 (PS1) phosphorylated at Ser367 specifically binds Annexin A2, which, through successive binding steps, facilitates this fusion. Lack of phosphorylation on PS1 1 Ser367 causes accumulation of partially fused autophagosomes and lysosomes in mouse brain and reduced autophagic flux. This inhibition of autophagy leads to decreased βCTF degradation and accumulation of toxic Aβ-peptide in the brain. This signaling pathway offers new potential drug targets for Alzheimer’s disease. Presenilin 1 (PS1), the catalytic subunit of the γ-secretase complex, cleaves βCTF to produce Aβ. We have shown that PS1 regulates Aβ levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 decreases Aβ levels by increasing βCTF degradation through autophagy. Here, we report the molecular mechanism by which PS1 modulates βCTF degradation. We show that PS1 phosphorylated at Ser367, but not nonphosphorylated PS1, interacts with Annexin A2, which, in turn, interacts with the lysosomal N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) Vamp8. Annexin A2 facilitates the binding of Vamp8 to the autophagosomal SNARE Syntaxin 17 to modulate the fusion of autophagosomes with lysosomes. Thus, PS1 phosphorylated at Ser367 has an antiamyloidogenic function, promoting autophagosome–lysosome fusion and increasing βCTF degradation. Drugs designed to increase the level of PS1 phosphorylated at Ser367 should be useful in the treatment of Alzheimer’s disease.

Bidirectional regulation of Aβ levels by Presenilin 1

Significance Alzheimer’s disease is the most common neurodegenerative disorder, affecting more than 5 million people in the United States. Multiple lines of evidence suggest that the accumulation of toxic oligomers and aggregates of β-amyloid (Aβ) peptide are the primary causes of neurodegeneration. Aβ originates from sequential cleavage of the amyloid precursor protein (APP). The APP first cleavage is by β-secretase and yields β-C-terminal fragment (βCTF). In turn, βCTF is cleaved by Presenilin 1 (PS1) to produce Aβ. In this work, we show that PS1, in addition to generating Aβ, can also decrease Aβ levels by directing βCTF degradation through autophagy. This previously unrecognized mechanism of regulation of Aβ by Presenilin 1 could provide an attractive target for potential Alzheimer’s disease therapies. Alzheimer’s disease (AD) is characterized by accumulation of the β-amyloid peptide (Aβ), which is generated through sequential proteolysis of the amyloid precursor protein (APP), first by the action of β-secretase, generating the β-C-terminal fragment (βCTF), and then by the Presenilin 1 (PS1) enzyme in the γ-secretase complex, generating Aβ. γ-Secretase is an intramembranous protein complex composed of Aph1, Pen2, Nicastrin, and Presenilin 1. Although it has a central role in the pathogenesis of AD, knowledge of the mechanisms that regulate PS1 function is limited. Here, we show that phosphorylation of PS1 at Ser367 does not affect γ-secretase activity, but has a dramatic effect on Aβ levels in vivo. We identified CK1γ2 as the endogenous kinase responsible for the phosphorylation of PS1 at Ser367. Inhibition of CK1γ leads to a decrease in PS1 Ser367 phosphorylation and an increase in Aβ levels in cultured cells. Transgenic mice in which Ser367 of PS1 was mutated to Ala, show dramatic increases in Aβ peptide and in βCTF levels in vivo. Finally, we show that this mutation impairs the autophagic degradation of βCTF, resulting in its accumulation and increased levels of Aβ peptide and plaque load in the brain. Our results demonstrate that PS1 regulates Aβ levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 also decreases Aβ levels by increasing βCTF degradation through autophagy. Elucidation of the mechanism by which PS1 regulates βCTF degradation may aid in the development of potential therapies for Alzheimer’s disease.

Gleevec shifts APP processing from a β-cleavage to a nonamyloidogenic cleavage

Significance Alzheimer’s disease (AD) is the leading cause of dementia in the elderly. Four approved drugs are used to treat AD cognitive symptoms, including loss of short-term memory. These drugs produce modest, temporary benefits at best and do not prevent or delay worsening of the disease. Recently, a mutation that protects elderly people from developing AD was discovered. The cellular process responsible for the mutation’s protective effect was also identified, suggesting that drugs targeting this process or pathway also might provide protection against the development of AD. In the present study, we discovered that the anticancer drug Gleevec and a related compound mimic the effects of the protective mutation and thus can act as models for the development of effective drugs to fight AD. Neurotoxic amyloid-β peptides (Aβ) are major drivers of Alzheimer’s disease (AD) and are formed by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (BACE) and γ-secretase. Our previous study showed that the anticancer drug Gleevec lowers Aβ levels through indirect inhibition of γ-secretase activity. Here we report that Gleevec also achieves its Aβ-lowering effects through an additional cellular mechanism. It renders APP less susceptible to proteolysis by BACE without inhibiting BACE enzymatic activity or the processing of other BACE substrates. This effect closely mimics the phenotype of APP A673T, a recently discovered mutation that protects carriers against AD and age-related cognitive decline. In addition, Gleevec induces formation of a specific set of APP C-terminal fragments, also observed in cells expressing the APP protective mutation and in cells exposed to a conventional BACE inhibitor. These Gleevec phenotypes require an intracellular acidic pH and are independent of tyrosine kinase inhibition, given that a related compound lacking tyrosine kinase inhibitory activity, DV2-103, exerts similar effects on APP metabolism. In addition, DV2-103 accumulates at high concentrations in the rodent brain, where it rapidly lowers Aβ levels. This study suggests that long-term treatment with drugs that indirectly modulate BACE processing of APP but spare other BACE substrates and achieve therapeutic concentrations in the brain might be effective in preventing or delaying the onset of AD and could be safer than nonselective BACE inhibitor drugs.