CJ1

Amino Acid Sequence

3D Structure (/structures/AF-P53355-F1-model_v6.cif)

Function

Calcium/calmodulin-dependent serine/threonine kinase involved in multiple cellular signaling pathways that trigger cell survival, apoptosis, and autophagy. Regulates both type I apoptotic and type II autophagic cell deaths signal, depending on the cellular setting. The former is caspase-dependent, while the latter is caspase-independent and is characterized by the accumulation of autophagic vesicles. Phosphorylates PIN1 resulting in inhibition of its catalytic activity, nuclear localization, and cellular function. Phosphorylates TPM1, enhancing stress fiber formation in endothelial cells. Phosphorylates STX1A and significantly decreases its binding to STXBP1. Phosphorylates PRKD1 and regulates JNK signaling by binding and activating PRKD1 under oxidative stress. Phosphorylates BECN1, reducing its interaction with BCL2 and BCL2L1 and promoting the induction of autophagy. Phosphorylates TSC2, disrupting the TSC1-TSC2 complex and stimulating mTORC1 activity in a growth factor-dependent pathway. Phosphorylates RPS6, MYL9 and DAPK3. Acts as a signaling amplifier of NMDA receptors at extrasynaptic sites for mediating brain damage in stroke. Cerebral ischemia recruits DAPK1 into the NMDA receptor complex and it phosphorylates GRINB at Ser-1303 inducing injurious Ca2+ influx through NMDA receptor channels, resulting in an irreversible neuronal death. Required together with DAPK3 for phosphorylation of RPL13A upon interferon-gamma activation which is causing RPL13A involvement in transcript-selective translation inhibition. Isoform 2 cannot induce apoptosis but can induce membrane blebbing.

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

We conducted a pharmacological inhibition study to further investigate the mechanism underlying the CJ1-mediated regulation of DAPK1 protein stability. SH-SY5Y cells were pretreated with the proteasome inhibitor MG-132 to block proteolytic activity, followed by the administration of CJ1. The inhibition of proteasomal activity by MG-132 prevented the CJ1-induced degradation of DAPK1, whereas a significant decrease in DAPK1 levels was observed in DMSO-treated controls under the same conditions (Fig. 3H and I). To further determine whether CJ1 enhances DAPK1 ubiquitination in cells, we conducted ubiquitination assays in which DAPK1 and HA-ubiquitin were co-transfected, following CJ1 treatment. In the presence of CJ1, DAPK1 polyubiquitination was markedly increased (Fig. 3J and K). These findings indicate that CJ1 promotes DAPK1 degradation by ubiquitin-dependent manner.

Description

In primary cortical neurons, the half-maximal degradation concentration (DC50) of CJ1, which represents the concentration required to achieve the degradation of 50 % of DAPK1, was determined to be 0.1842 μM.

Description

A CCK-8 assay was performed using mouse primary cortical neurons to evaluate the cytotoxicity of CJ1. The results showed that CJ1 did not significantly affect cell viability across a concentration range of 0.1 to 20 μM, suggesting a low potential for cytotoxicity (Fig. 3A).