Protein kinase RNA-activated also known asprotein kinase R (PKR),interferon-induced, double-stranded RNA-activated protein kinase, oreukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) is anenzyme that in humans is encoded by theEIF2AK2gene on chromosome 2.[5][6] PKR is a serine/tyrosinekinase that is 551 amino acids long.[7]
PKR is inducible by various mechanisms of stress and protects againstviral infections.[8] It also has a role in several signaling pathways.[9][10]
Protein kinase-R is activated bydouble-stranded RNA (dsRNA), introduced to the cells by a viral infection.[9] In situations of viral infection, the dsRNA created by viral replication and gene expression binds to theN-terminal domain, activating the protein.[9] PKR activation via dsRNA is length dependent, requiring the dsRNA to be 30 bp in length to bind to PKR molecules.[9] However, excess dsRNA can diminish activation of PKR.[9] Binding to dsRNA is believed to activate PKR by inducingdimerization of the kinase domains and subsequent auto-phosphorylation reactions.[9] It is not yet established whether PKR activates in cis, with a protomer's activation loop reaching into its own catalytic site, or in trans, with the activation loop being phosphorylated in a face to face geometry by a conjugate protomer.[11] PKR can also be activated by the proteinPACT via phosphorylation of S287 on its M3 domain.[12] Thepromoter region of PKR has interferon-stimulated response elements to whichType I interferons (IFN) bind to induce the transcription of PKR genes.[12][13] Some research suggests that PKR can be stimulated byheat shock proteins,heparin,growth factors, bacterial infection, pro-inflammatory cytokines,reactive oxygen species, DNA damage, mechanical stress, and excess nutrient intake.[12]
Once active, PKR is able to phosphorylate the eukaryotic translation initiation factoreIF2α.[12] This inhibits further cellular mRNA translation, thereby preventing viral protein synthesis.[10] Overall, this leads to apoptosis of virally infected cells to prevent further viral spread. PKR can also induceapoptosis in bacterial infection by responding to LPS and proinflammatory cytokines.[10] Apoptosis can also occur via PKR activation of theFADD andcaspase signaling pathway.[13]
PKR also has pro-inflammatory functions, as it can mediate the activation of the transcription factorNF-kB, by phosphorylating its inhibitory subunit, IkB.[13] This leads to the expression ofadhesion molecules and transcription factors that activate them, which induce inflammation responses such as the secretion of pro-inflammatory cytokines.[12] PKR also activates severalmitogen-activated protein kinases (MAPK) to lead to inflammation.[13]
To balance the effects of apoptosis and inflammation, PKR has regulatory functions. Active PKR is also able to activate tumor suppressorPP2A which regulates the cell cycle and the metabolism.[14] There is also evidence that PKR isautophagic as a regulatory mechanism.[13]
Figure showing the different signaling pathways that activated PKR plays a role in. Most results of these pathways help in fighting off viral infection and regulating the immune response, conferring PKR with apoptotic and pro-inflammatory functionality.
PKR is in the center of cellular response to different stress signals such as pathogens, lack of nutrients, cytokines,irradiation, mechanical stress, orER stress.[12] The PKR pathway leads to a stress response through activation of other stress pathways such asJNK,p38,NFkB,PP2A and phosphorylation ofeIF2α.[10] ER stress caused by excess of unfolded proteins leads to inflammatory responses.[15] PKR contributes to this response by interacting with several inflammatory kinases such as IKK, JNK, ElF2α,insulin receptors and others.[15] This metabolically activated inflammatory complex is called metabolic inflammasome or metaflammasome.[16][17] Via the JNK signaling pathway, PKR also plays a role in insulin resistance, diabetes, and obesity by phosphorylatingIRS1.[18] Inhibiting PKR in mice led to lower inflammation in adipose tissues, increased sensitivity to insulin, and amelioration of diabetic symptoms.[18] PKR also participates in themitochondrial unfolded protein response (UPRmt).[19] Here, PKR is induced via the transcription factorAP-1 and activated independently of PACT.[19] In this context, PKR has been shown to be relevant to intestinal inflammation.[19]
Viruses have developed many mechanisms to counteract the PKR mechanism. It may be done byDecoy dsRNA, degradation, hiding of viraldsRNA, dimerization block, dephosphorylation of substrate or by apseudosubstrate. Some mechanisms are still unknown, for instance, inLymphocytic choriomeningitis virus infection the virus is not recognized by PKR,[20] and the current anti-dsRNA antibodies have limitation in detectingdsRNA species of negative stranded RNA viruses,[21] which necessitates the use of a different approach to understand this viral defense mechanism.
First report in 2002 has been shown thatimmunohistochemical marker for phosphorylated PKR and eIF2α was displayed positively in degenerating neurons in the hippocampus and the frontal cortex of patients withAlzheimer's disease (AD), suggesting the link between PKR and AD. Additionally, many of these neurons were also immunostained with an antibody for phosphorylatedTau protein.[24] Activated PKR was specifically found in the cytoplasm and nucleus, as well as co-localized with neuronal apoptotic markers.[25] Further studies have assessed the levels of PKR in blood andcerebrospinal fluid (CSF) of AD patients and controls. The result of an analysis of the concentrations of total and phosphorylated PKR (pPKR) inperipheral blood mononuclear cells (PBMCs) in 23 AD patients and 19 control individuals showed statistically significant increased levels of the ratio of phosphorylated PKR/PKR in AD patients compared with controls.[26] Assessments of CSF biomarkers, such asAβ1-42,Aβ1-40, Tau, and phosphorylated Tau at threonine 181, have been a validated use in clinical research and in routine practice to determine whether patients have CSF abnormalities and AD brain lesions. A study found that "total PKR and pPKR concentrations were elevated in AD and amnestic mild cognitive impairment subjects with a pPKR value (optical density units) discriminating AD patients from control subjects with a sensitivity of 91.1% and a specificity of 94.3%. Among AD patients, total PKR and pPKR levels correlate with CSF p181tau levels. Some AD patients with normal CSF Aß, T-tau, or p181tau levels had abnormal total PKR and pPKR levels".[27] It was concluded that the PKR-eIF2α pro-apoptotic pathway could be involved inneuronal degeneration that leads to various neuropathological lesions as a function of neuronal susceptibility.
PKR and beta amyloid
Activation of PKR can cause accumulation ofamyloid β-peptide (Aβ) via de-repression ofBACE1 (β-site APP Cleaving Enzyme) expression in Alzheimer Disease patients.[28] Normally, the5′ untranslated region (5′ UTR) in the BACE1 promoter would fundamentally inhibit the expression of BACE1 gene. However, BACE1 expression can be activated by phosphorylation of eIF2a, which reverses the inhibitory effect exerted by BACE1 5′ UTR. Phosphorylation of eIF2a is triggered by activation of PKR. Viral infection such asherpes simplex virus (HSV) or oxidative stress can both increase BACE1 expression through activation of PKR-eIF2a pathway.[29]
In addition, the increased activity of BACE1 could also lead to β-cleaved carboxy-terminal fragment of β-Amyloid precursor protein (APP-βCTF) induced dysfunction ofendosomes in AD.[30] Endosomes are highly active β-Amyloid precursor protein (APP) processing sites, and endosome abnormalities are associated with upregulated expression of early endosomal regulator,Rab5. These are the earliest known disease-specific neuronal response in AD. Increased activity of BACE1 leads to synthesis of the APP-βCTF. An elevated level of βCTF then causes Rab5 overactivation. βCTF recruitsAPPL1 to rab5 endosomes, where it stabilizes activeGTP-Rab5, leading to pathologically accelerated endocytosis, endosome swelling and selectively impaired axonal transport of Rab5 endosomes.
PKR and Tau phosphorylation
It is reported earlier that phosphorylated PKR could co-localize with phosphorylated Tau protein in affected neurons.[31][24] A proteinphosphatase-2A inhibitor (PP2A inhibitor) –okadaic acid (OA) – is known to increase tau phosphorylation, Aβ deposition and neuronal death. It is studied that OA also induces PKR phosphorylation and thus, eIF2a phosphorylation. eIF2a phosphorylation then induces activation of transcription factor 4 (ATF4), which inducesapoptosis and nuclear translocation, contributing to neuronal death.[32]
Glycogen synthase kinase 3β (GSK-3β) is responsible for tau phosphorylation and controls several cellular functions including apoptosis. Another study demonstrated thattunicamycin or Aβ treatment can induce PKR activation in humanneuroblastoma cells and can trigger GSK3β activation, as well as tau phosphorylation. They found that in AD brains, both activated PKR and GSK3β co-localize with phosphorylated tau in neurons. In SH-SY5Y cell cultures, tunicamycin and Aβ(1-42) activate PKR, which then can modulate GSK-3β activation and induce tau phosphorylation, apoptosis. All these processes are attenuated by PKR inhibitors or PKRsiRNA. PKR could represent a crucial signaling point relaying stress signals to neuronal pathways by interacting withtranscription factor or indirectly controlling GSK3β activation, leading to cellular degeneration in AD.[33]
^Gupta P, Taiyab A, Hassan MI (January 2021). Donev R (ed.). "Emerging role of protein kinases in diabetes mellitus: From mechanism to therapy".Advances in Protein Chemistry and Structural Biology. Protein Kinases in Drug Discovery.124. Academic Press:47–85.doi:10.1016/bs.apcsb.2020.11.001.ISBN9780323853132.PMID33632470.S2CID229608384.
^abChang RC, Wong AK, Ng HK, Hugon J (December 2002). "Phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha) is associated with neuronal degeneration in Alzheimer's disease".NeuroReport.13 (18):2429–2432.doi:10.1097/00001756-200212200-00011.PMID12499843.S2CID84266563.
^Page G, Rioux Bilan A, Ingrand S, Lafay-Chebassier C, Pain S, Perault Pochat MC, et al. (2006). "Activated double-stranded RNA-dependent protein kinase and neuronal death in models of Alzheimer's disease".Neuroscience.139 (4):1343–1354.doi:10.1016/j.neuroscience.2006.01.047.PMID16581193.S2CID36700744.
^Paccalin M, Pain-Barc S, Pluchon C, Paul C, Besson MN, Carret-Rebillat AS, et al. (2006). "Activated mTOR and PKR kinases in lymphocytes correlate with memory and cognitive decline in Alzheimer's disease".Dementia and Geriatric Cognitive Disorders.22 (4):320–326.doi:10.1159/000095562.PMID16954686.S2CID45647507.
^Mouton-Liger F, Paquet C, Dumurgier J, Lapalus P, Gray F, Laplanche JL, et al. (May 2012). "Increased cerebrospinal fluid levels of double-stranded RNA-dependant protein kinase in Alzheimer's disease".Biological Psychiatry.71 (9):829–835.doi:10.1016/j.biopsych.2011.11.031.PMID22281122.S2CID21131086.
^Peel AL, Bredesen DE (October 2003). "Activation of the cell stress kinase PKR in Alzheimer's disease and human amyloid precursor protein transgenic mice".Neurobiology of Disease.14 (1):52–62.doi:10.1016/S0969-9961(03)00086-X.PMID13678666.S2CID13109874.
^Kim SM, Yoon SY, Choi JE, Park JS, Choi JM, Nguyen T, et al. (September 2010). "Activation of eukaryotic initiation factor-2 α-kinases in okadaic acid-treated neurons".Neuroscience.169 (4):1831–1839.doi:10.1016/j.neuroscience.2010.06.016.PMID20600673.S2CID207248721.
^Langland JO, Kao PN, Jacobs BL (May 1999). "Nuclear factor-90 of activated T-cells: A double-stranded RNA-binding protein and substrate for the double-stranded RNA-dependent protein kinase, PKR".Biochemistry.38 (19):6361–6368.doi:10.1021/bi982410u.PMID10320367.
^Gil J, Esteban M, Roth D (December 2000). "In vivo regulation of the dsRNA-dependent protein kinase PKR by the cellular glycoprotein p67".Biochemistry.39 (51):16016–16025.doi:10.1021/bi001754t.PMID11123929.
Thomis DC, Doohan JP, Samuel CE (May 1992). "Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells".Virology.188 (1):33–46.doi:10.1016/0042-6822(92)90732-5.PMID1373553.
McCormack SJ, Thomis DC, Samuel CE (May 1992). "Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase".Virology.188 (1):47–56.doi:10.1016/0042-6822(92)90733-6.PMID1373554.
Mellor H, Proud CG (July 1991). "A synthetic peptide substrate for initiation factor-2 kinases".Biochemical and Biophysical Research Communications.178 (2):430–437.doi:10.1016/0006-291X(91)90125-Q.PMID1677563.
Meurs E, Chong K, Galabru J, Thomas NS, Kerr IM, Williams BR, et al. (July 1990). "Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon".Cell.62 (2):379–390.doi:10.1016/0092-8674(90)90374-N.PMID1695551.S2CID20477995.
Silverman RH, Sengupta DN (1991). "Translational regulation by HIV leader RNA, TAT, and interferon-inducible enzymes".Journal of Experimental Pathology.5 (2):69–77.PMID1708818.
Roy S, Katze MG, Parkin NT, Edery I, Hovanessian AG, Sonenberg N (March 1990). "Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product".Science.247 (4947):1216–1219.Bibcode:1990Sci...247.1216R.doi:10.1126/science.2180064.PMID2180064.
Barber GN, Edelhoff S, Katze MG, Disteche CM (June 1993). "Chromosomal assignment of the interferon-inducible double-stranded RNA-dependent protein kinase (PRKR) to human chromosome 2p21-p22 and mouse chromosome 17 E2".Genomics.16 (3):765–767.doi:10.1006/geno.1993.1262.PMID7686883.
Squire J, Meurs EF, Chong KL, McMillan NA, Hovanessian AG, Williams BR (June 1993). "Localization of the human interferon-induced, ds-RNA activated p68 kinase gene (PRKR) to chromosome 2p21-p22".Genomics.16 (3):768–770.doi:10.1006/geno.1993.1263.PMID7686884.
Kuhen KL, Shen X, Carlisle ER, Richardson AL, Weier HU, Tanaka H, et al. (August 1996). "Structural organization of the human gene (PKR) encoding an interferon-inducible RNA-dependent protein kinase (PKR) and differences from its mouse homolog".Genomics.36 (1):197–201.doi:10.1006/geno.1996.0446.PMID8812437.
Kuhen KL, Shen X, Samuel CE (October 1996). "Mechanism of interferon action sequence of the human interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones".Gene.178 (1–2):191–193.doi:10.1016/0378-1119(96)00314-9.PMID8921913.
