AbH6

Amino Acid Sequence

3D Structure (/structures/AF-P00519-F1-model_v6.cif)

Function

Non-receptor tyrosine-protein kinase that plays a role in many key processes linked to cell growth and survival such as cytoskeleton remodeling in response to extracellular stimuli, cell motility and adhesion, receptor endocytosis, autophagy, DNA damage response and apoptosis. Coordinates actin remodeling through tyrosine phosphorylation of proteins controlling cytoskeleton dynamics like WASF3 (involved in branch formation); ANXA1 (involved in membrane anchoring); DBN1, DBNL, CTTN, RAPH1 and ENAH (involved in signaling); or MAPT and PXN (microtubule-binding proteins). Phosphorylation of WASF3 is critical for the stimulation of lamellipodia formation and cell migration. Involved in the regulation of cell adhesion and motility through phosphorylation of key regulators of these processes such as BCAR1, CRK, CRKL, DOK1, EFS or NEDD9. Phosphorylates multiple receptor tyrosine kinases and more particularly promotes endocytosis of EGFR, facilitates the formation of neuromuscular synapses through MUSK, inhibits PDGFRB-mediated chemotaxis and modulates the endocytosis of activated B-cell receptor complexes. Other substrates which are involved in endocytosis regulation are the caveolin (CAV1) and RIN1. Moreover, ABL1 regulates the CBL family of ubiquitin ligases that drive receptor down-regulation and actin remodeling. Phosphorylation of CBL leads to increased EGFR stability. Involved in late-stage autophagy by regulating positively the trafficking and function of lysosomal components. ABL1 targets to mitochondria in response to oxidative stress and thereby mediates mitochondrial dysfunction and cell death. In response to oxidative stress, phosphorylates serine/threonine kinase PRKD2 at 'Tyr-717'. ABL1 is also translocated in the nucleus where it has DNA-binding activity and is involved in DNA-damage response and apoptosis. Many substrates are known mediators of DNA repair: DDB1, DDB2, ERCC3, ERCC6, RAD9A, RAD51, RAD52 or WRN. Activates the proapoptotic pathway when the DNA damage is too severe to be repaired. Phosphorylates TP73, a primary regulator for this type of damage-induced apoptosis. Phosphorylates the caspase CASP9 on 'Tyr-153' and regulates its processing in the apoptotic response to DNA damage. Phosphorylates PSMA7 that leads to an inhibition of proteasomal activity and cell cycle transition blocks. ABL1 also acts as a regulator of multiple pathological signaling cascades during infection. Several known tyrosine-phosphorylated microbial proteins have been identified as ABL1 substrates. This is the case of A36R of Vaccinia virus, Tir (translocated intimin receptor) of pathogenic E.coli and possibly Citrobacter, CagA (cytotoxin-associated gene A) of H.pylori, or AnkA (ankyrin repeat-containing protein A) of A.phagocytophilum. Pathogens can highjack ABL1 kinase signaling to reorganize the host actin cytoskeleton for multiple purposes, like facilitating intracellular movement and host cell exit. Finally, functions as its own regulator through autocatalytic activity as well as through phosphorylation of its inhibitor, ABI1. Regulates T-cell differentiation in a TBX21-dependent manner (By similarity). Positively regulates chemokine-mediated T-cell migration, polarization, and homing to lymph nodes and immune-challenged tissues, potentially via activation of NEDD9/HEF1 and RAP1 (By similarity). Phosphorylates TBX21 on tyrosine residues leading to an enhancement of its transcriptional activator activity (By similarity).

Amino Acid Sequence

3D Structure (/structures/struct-20261026-093601-422329.cif)

Function

BCR-ABL1 encodes a tyrosine kinase located in the cytoplasm which has constitutive activity. The BCR-ABL1 kinase continuously activates several signaling pathways such as Ras-mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and signal transducer and activator of transcription 5 (STAT-5), resulting in increased cell proliferation and survival. But BCR-ABL1 does not just kickstart cellular transformation; rather, leukemic cells are dependent on BCR-ABL1 for their survival. BCR-ABL1 is thus a classic example of oncogene addiction.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

To elucidate the target degradation mechanism, we first conducted rescue assays (Figure 4J and Figure S5 ). Pretreatment of K562 cells with MG132 (a proteasome inhibitor) effectively reversed AbH6 -induced degradation of endogenous BCRABL/ABL, whereas chloroquine (a lysosome inhibitor) had no such effect, indicating that AbH6 triggered targeted degradation through the ubiquitin-proteasome system (UPS) rather than the autophagy-lysosome pathway [ 1–4 ]. In contrast to the reported PROTAC-based degrader, SIAIS056 [ 50 ], MLN4924 (inhibitor of NEDD8-activating enzyme) failed to block BCR-ABL/ABL degradation by AbH6 , suggesting its distinct degradation process, likely operated independently of neddylation-dependent E3 ligase activity [ 51 ]. To further investigate the involvement of molecular chaperones, K562 cells were pretreated with either VER-155008 or Geldanamycin prior to AbH6 treatment. At the concentrations initially used, both inhibitors effectively blocked AbH6-induced ABL1 degradation, suggesting the involvement of the HSP70– HSP90 chaperone system in this process. Notably, when lower concentrations of the inhibitors were applied, ABL1 degradation was partially restored, indicating that strong inhibition of the chaperone machinery is required to fully suppress AbH6mediated degradation. This hypothesis was strengthened by co-immunoprecipitation (Co-IP) experiments (Figure 4K ); results revealed that, in AbH6 -treated K562 cells, both target ubiquitination and its association with HSP70/90 occurred. Collectively, these data clearly establish a target degradation mechanism for AbH6 -induced degradation of BCR-ABL/ABL, which might be more broadly applicable to other HyT degraders (Figure 1C ). We propose that both HSP70 and HSP90 may participate in distinct stages of the HyT-bound protein target, with HSP70 primarily facilitating early-stage folding events while HSP90 takes part in the maturation of late-stage folding intermediates [ 52 ]. This chaperone-mediated mechanism causes protein misfolding and subsequent ubiquitination, thus engaging UPS and leading to eventual target degradation without requiring the recruitment of canonical E3 ligases.

Description

among them, AbH6 emerged as the most robust HyT-based degrader, capable of degrading both endogenous BCR-ABL and ABL from K562 cells in both concentration- and time-dependent manners (DC50 = 185 ± 29 nM and 210 ± 40 nM, respectively; Figure 4C–E )

Description

among them, AbH6 emerged as the most robust HyT-based degrader, capable of degrading both endogenous BCR-ABL and ABL from K562 cells in both concentration- and time-dependent manners (DC50 = 185 ± 29 nM and 210 ± 40 nM, respectively; Figure 4C–E ).

Description

All HyTs also displayed potent and highly selective antiproliferative activity in BCRABL-expressing K562 cells (Figure 4B and Figure S3B ). Among them, AbH4 displayed the best in vitro kinase inhibition (IC50 = 18 ± 7 nM), while AbH6 demonstrated the best antiproliferative activity (GI50 = 102 ± 3 nM; Figure 4E ). AbH6 also potently inhibited proliferation of Ba/F3 cells expressing the BCR-ABLT315I mutant (GI50 = 107 ± 2 nM)

Description

All HyTs also displayed potent and highly selective antiproliferative activity in BCRABL-expressing K562 cells (Figure 4B and Figure S3B ). Among them, AbH4 displayed the best in vitro kinase inhibition (IC50 = 18 ± 7 nM), while AbH6 demonstrated the best antiproliferative activity (GI50 = 102 ± 3 nM; Figure 4E ). AbH6 also potently inhibited proliferation of Ba/F3 cells expressing the BCR-ABLT315I mutant (GI50 = 107 ± 2 nM)

Description

FIGURE 4 (A) In vitro kinase inhibition (IC50 ) plot of various ABL-degrading HyTs against full-length ABL1 kinase (1-h incubation).  (E) Summary of IC50 , GI50 , and DC50 values of various ABL-degrading HyTs in K562 and BaF3T315I cells