Study On The Function Of Protein Kinase D1 In Endoplasmic Reticulum Stress Response

As an important organelle in eukaryocyte,endoplasmic reticulum?ER?,is responsible for synthesis,posttranslational modification and trafficking of secretory and transmembrane proteins,as well as responsible for calcium storage and lipid synthesis.Acumulation of unfolded proteins in the ER lumen alters ER functions and induces a cellular change known as ER stress,and further initiates the unfolded protein response?UPR?.UPR is mediated by three different ER stress sensors:inositol-requiring enzyme 1?IRE1?,protein kinase R?PKR?-like endoplasmic reticulum kinase?PERK?and activating transcription factor 6?ATF6?.UPR is triggered to relieve ER stress and restore ER homeostasis.When UPR fails,apoptosis will be induced to remove the severely damaged cells.ER stress is involved in various diseases,including neurondegenerative disease and cancer.However,the mechanisms underlying cell survival and death upon ER stress still remain unclear.As a serine threonine kinase family,protein kinase D?PKD?belongs to Ca²⁺/calmodulin-dependent protein kinase?CAMK?super family,consisting of three sub-members PKD1,2,and 3.Increasing evidences support that PKD1 plays an important role in a number of cellular processes,such as proliferation,differenciation,apoptosis,protein secretion,as well as diverse pathological diseases,including cardiac diseases and tumor.It has been reported that PKD1 could initiate protective signaling against oxidative stress,condition impinging on ER stress induction.However,there is no study reporting the direct activation of PKD1 during ER stress.Here,we focus on exploring the roles of PKD1 in ER stress response,as well as investigating the binding mode of ATP non-competitive PKD inhibitors.At first,we demonstrated that ER stress could induce PKD1 activation through PLC/PKC?pathway in HepG2 and PC-3 cells.Based on the results from TUNEL and MTT assay,we found that knockdown or inhibition of PKD1 would increase cell sensitivity to ER stress,as shown by the increase of cleaved caspase-3 and PARP and increase of cell apoptotic ratio.And then we figured out that PKD1 specifically regulated IRE1 signaling pathway under ER stress.PKD1 siRNAs or PKD inhibitor selectively downregulated IRE1 protein level,without changing IRE1 mRNA expression and translation.MG132,a specific proteasome inhibitor,could reverse the decrease of IRE1protein induced by PKD1 inhibition,which suggested that PKD1 regulates IRE1 protein stability.In consistent with this,we further found that PKD1 promoted IRE1/XBP1signaling pathway through enhancing IRE1 protein stability.IRE1/XBP1 overexpression could decrease cell apoptosis induced by PKD inhibition under ER stress,which means PKD1 promotes cell resistance to ER tress through IRE1/XBP1 pathway.Further,we discovered that activation of PKD1 inhibits the hyperactivation of IRE1/JNK pathway induced by ER stress through MPK1.When PKD1 was knocked down or inhibited,the phosphorylation of MKP1 induced by ER stress was largely blocked,which leading to the switch of JNK activation from transient model to longtime-lasting and hyperactive pattern.Meanwhile,MKP1 inhibition also leads to increased cell apoptosis induced by ER stress.All results above indicated that PKD1 protects cells from ER stress-induced apoptosis through two ways,the first is maintaining the protein stability of IRE1 and enhancing the activity of IRE1/XBP1s pathway during ER stress to promote the protective function of IRE1;the other way is preventing the hyperactivation of IRE1/JNK pathway via MKP1and inhibiting the deleterious role of IRE1 under ER stress condition.PKD has been demonstrated to play an important role in cardiomyopathy and cancer,which makes PKD a hot spot for drug development.A dozen of PKD inhibitors have been discovered via either high throughput screening or chemical modification tests.However,the residues responsible for the binding of inhibitors in the kinase domain are far from clear.In this study,we investigated the working model of two ATP non-competitive inhibitors of PKD1,kb-NB 142-70?kb?and CID755673?CID?,through homologous modeling,molecular docking and site-directed mutagenesis.Our results indicated that Leu⁶⁶²,Leu⁷¹³ and Phe⁷²⁸ in PKD1 kinase domain were the key residues for kb/CID binding,and the docking of ATP in the kinase domain was required for the stable binding of kb/CID.Hydrophobic interactions provided by Leu⁶⁶²,Leu⁷¹³ and Phe⁷²⁸ in kinase domain of PKD are involved in the regulation of the inhibitory efficiency of kb/CID.Meanwhile,replacement of Cys⁷²⁶ in the kinase domain of PKD1 with Ala resulted in reduced inhibition activity of kb toward PKD,but had no impact on the activity of CID.We speculated that unique interaction with Cys⁷²⁶ may account for the higher potency of kb over CID.In brief,our work strongly proved that PKD1 is a tight regulator of IRE1.PKD1protects cell from ER stress-induced apoptosis by promoting prosurvival IRE1 signaling and attenuating IRE1-dependent proapoptotic JNK activity.This study clearly indicates that PKD1 plays an essencial role in UPR mediated cell fate decision,which would expand our understanding of ER stress response in related physiologic and pathologic processes.Our study also revealed the amino acid network interacting with ATP non-competitive inhibitors in the activation pocket of PKD1,which would contribute to the rational design of PKD inhibitors with valuable therapeutic advantages.