HY12

Amino Acid Sequence

3D Structure (/structures/AF-P45983-F1-model_v6.cif)

Function

Serine/threonine-protein kinase involved in various processes such as cell proliferation, differentiation, migration, transformation and programmed cell death. Extracellular stimuli such as pro-inflammatory cytokines or physical stress stimulate the stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) signaling pathway. In this cascade, two dual specificity kinases MAP2K4/MKK4 and MAP2K7/MKK7 phosphorylate and activate MAPK8/JNK1. In turn, MAPK8/JNK1 phosphorylates a number of transcription factors, primarily components of AP-1 such as JUN, JDP2 and ATF2 and thus regulates AP-1 transcriptional activity. Phosphorylates the replication licensing factor CDT1, inhibiting the interaction between CDT1 and the histone H4 acetylase HBO1 to replication origins. Loss of this interaction abrogates the acetylation required for replication initiation. Promotes stressed cell apoptosis by phosphorylating key regulatory factors including p53/TP53 and Yes-associates protein YAP1. In T-cells, MAPK8 and MAPK9 are required for polarized differentiation of T-helper cells into Th1 cells. Contributes to the survival of erythroid cells by phosphorylating the antagonist of cell death BAD upon EPO stimulation. Mediates starvation-induced BCL2 phosphorylation, BCL2 dissociation from BECN1, and thus activation of autophagy. Phosphorylates STMN2 and hence regulates microtubule dynamics, controlling neurite elongation in cortical neurons (By similarity). In the developing brain, through its cytoplasmic activity on STMN2, negatively regulates the rate of exit from multipolar stage and of radial migration from the ventricular zone (By similarity). Phosphorylates several other substrates including heat shock factor protein 4 (HSF4), the deacetylase SIRT1, ELK1, or the E3 ligase ITCH. Phosphorylates the CLOCK-BMAL1 heterodimer and plays a role in the regulation of the circadian clock. Phosphorylates the heat shock transcription factor HSF1, suppressing HSF1-induced transcriptional activity. Phosphorylates POU5F1, which results in the inhibition of POU5F1's transcriptional activity and enhances its proteasomal degradation (By similarity). Phosphorylates JUND and this phosphorylation is inhibited in the presence of MEN1. In neurons, phosphorylates SYT4 which captures neuronal dense core vesicles at synapses (By similarity). Phosphorylates EIF4ENIF1/4-ET in response to oxidative stress, promoting P-body assembly. Phosphorylates SIRT6 in response to oxidative stress, stimulating its mono-ADP-ribosyltransferase activity. Phosphorylates NLRP3, promoting assembly of the NLRP3 inflammasome. Phosphorylates ALKBH5 in response to reactive oxygen species (ROS), promoting ALKBH5 sumoylation and inactivation.

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 degradation mechanism, the proteasome inhibitor MG132 and the NEDD8-activating enzyme (NAE) inhibitor MLN4924 were used to disrupt the ubiquitin-proteasome system (UPS) function. Inhibition of the 26S proteasome with MG132 or inactivation of cullinRING ligases via MLN4924 attenuated HY12-induced JNK1 degradation (Fig. 6A), confirming UPS-dependence. Given the potential involvement of endogenous chaperones in degrading misfolded proteins, we further assessed the roles of Hsp70 and Hsp90. Pharmacological inhibition of Hsp70 with VER155008 or Hsp90 with 17-AAG failed to reverse HY12-mediated JNK1 degradation (Fig. 6C), indicating these chaperones are dispensable for this process. Collectively, these findings indicate that HY12 induces JNK1 degradation through both the UPS and autophagy-lysosome pathways.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

The role of the autophagy-lysosome pathway was then examined using 3-methyladenine (3-MA) to inhibit the early stage of autophagosome formation and hydroxychloroquine (HCQ) to block the late stage of autophagic degradation in lysosomes. Notably, HCQ significantly impaired HY12-induced JNK1 degradation, whereas 3-MA had no effect (Fig. 6B), suggesting that HY12 likely reaches the lysosome for degradation but acts independently of early autophagosome formation. Given the potential involvement of endogenous chaperones in degrading misfolded proteins, we further assessed the roles of Hsp70 and Hsp90. Pharmacological inhibition of Hsp70 with VER155008 or Hsp90 with 17-AAG failed to reverse HY12-mediated JNK1 degradation (Fig. 6C), indicating these chaperones are dispensable for this process. Collectively, these findings indicate that HY12 induces JNK1 degradation through both the UPS and autophagy-lysosome pathways.

Description

HY12 induced potent, concentration- and time-dependent degradation of JNK1 (DC50 = 0.16 μM in BEAS-2B cells).