Protein kinase R explained

Protein kinase RNA-activated also known as protein kinase R (PKR), interferon-induced, double-stranded RNA-activated protein kinase, or eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) is an enzyme that in humans is encoded by the EIF2AK2 gene on chromosome 2.^{[1] [2]} PKR is a serine/tyrosine kinase that is 551 amino acids long.^[3]

PKR is inducible by various mechanisms of stress and protects against viral infections.^[4] It also has a role in several signaling pathways.^{[5] [6]}

Mechanism of action

Protein kinase-R is activated by double-stranded RNA (dsRNA), introduced to the cells by a viral infection. In situations of viral infection, the dsRNA created by viral replication and gene expression binds to the N-terminal domain, activating the protein. PKR activation via dsRNA is length dependent, requiring the dsRNA to be 30 bp in length to bind to PKR molecules. However, excess dsRNA can diminish activation of PKR. Binding to dsRNA is believed to activate PKR by inducing dimerization of the kinase domains and subsequent auto-phosphorylation reactions. PKR can also be activated by the protein PACT via phosphorylation of S287 on its M3 domain.^[7] The promoter region of PKR has interferon-stimulated response elements to which Type I interferons (IFN) bind to induce the transcription of PKR genes.^[8] Some research suggests that PKR can be stimulated by heat shock proteins, heparin, growth factors, bacterial infection, pro-inflammatory cytokines, reactive oxygen species, DNA damage, mechanical stress, and excess nutrient intake.

Once active, PKR is able to phosphorylate the eukaryotic translation initiation factor eIF2α. This inhibits further cellular mRNA translation, thereby preventing viral protein synthesis. Overall, this leads to apoptosis of virally infected cells to prevent further viral spread. PKR can also induce apoptosis in bacterial infection by responding to LPS and proinflammatory cytokines. Apoptosis can also occur via PKR activation of the FADD and caspase signaling pathway.

PKR also has pro-inflammatory functions, as it can mediate the activation of the transcription factor NF-kB, by phosphorylating its inhibitory subunit, IkB. This leads to the expression of adhesion molecules and transcription factors that activate them, which induce inflammation responses such as the secretion of pro-inflammatory cytokines. PKR also activates several mitogen-activated protein kinases (MAPK) to lead to inflammation.

To balance the effects of apoptosis and inflammation, PKR has regulatory functions. Active PKR is also able to activate tumor suppressor PP2A which regulates the cell cycle and the metabolism.^[9] There is also evidence that PKR is autophagic as a regulatory mechanism.

PKR stress pathway

PKR is in the center of cellular response to different stress signals such as pathogens, lack of nutrients, cytokines, irradiation, mechanical stress, or ER stress. The PKR pathway leads to a stress response through activation of other stress pathways such as JNK, p38, NFkB, PP2A and phosphorylation of eIF2α. ER stress caused by excess of unfolded proteins leads to inflammatory responses.^[10] PKR contributes to this response by interacting with several inflammatory kinases such as IKK, JNK, ElF2α, insulin receptors and others. This metabolically activated inflammatory complex is called metabolic inflammasome or metaflammasome.^{[11] [12]} Via the JNK signaling pathway, PKR also plays a role in insulin resistance, diabetes, and obesity by phosphorylating IRS1.^[13] Inhibiting PKR in mice led to lower inflammation in adipose tissues, increased sensitivity to insulin, and amelioration of diabetic symptoms. PKR also participates in the mitochondrial unfolded protein response (UPR^mt). Here, PKR is induced via the transcription factor AP-1 and activated independently of PACT.^[14] In this context, PKR has been shown to be relevant to intestinal inflammation.

Viral defense

Viruses have developed many mechanisms to counteract the PKR mechanism. It may be done by Decoy dsRNA, degradation, hiding of viral dsRNA, dimerization block, dephosphorylation of substrate or by a pseudosubstrate.

For instance, Epstein–Barr virus (EBV) uses the gene EBER1 to produce decoy dsRNA. This leads to cancers such as Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and various leukemias.

Viral defense mechanisms against PKR

Defense type	Virus	Molecule
Decoy dsRNA		VAI RNA
		EBER
		TAR
PKR degradation		2A^pro
Hide viral dsRNA		E3L
		σ3
		NS1
Dimerization block	Influenza virus	p58^IPK
		NS5A
	Vaccinia virus	K3L
	HIV	Tat
Dephosphorylation of substrate		ICP34.5

Memory and learning

PKR knockout mice or inhibition of PKR in mice enhances memory and learning.^[15]

Neuronal degeneration disease

First report in 2002 has been shown that immunohistochemical marker for phosphorylated PKR and eIF2α was displayed positively in degenerating neurons in the hippocampus and the frontal cortex of patients with Alzheimer's disease (AD), suggesting the link between PKR and AD. Additionally, many of these neurons were also immunostained with an antibody for phosphorylated Tau protein.^[16] Activated PKR was specifically found in the cytoplasm and nucleus, as well as co-localized with neuronal apoptotic markers.^[17] Further studies have assessed the levels of PKR in blood and cerebrospinal fluid (CSF) of AD patients and controls. The result of an analysis of the concentrations of total and phosphorylated PKR (pPKR) in peripheral 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.^[18] Assessments of CSF biomarkers, such as Aβ1-42, 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".^[19] It was concluded that the PKR-eIF2α pro-apoptotic pathway could be involved in neuronal degeneration that leads to various neuropathological lesions as a function of neuronal susceptibility.

PKR and beta amyloid

Activation of PKR can cause accumulation of amyloid β-peptide (Aβ) via de-repression of BACE1 (β-site APP Cleaving Enzyme) expression in Alzheimer Disease patients.^[20] Normally, the 5′ 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 as herpes simplex virus (HSV) or oxidative stress can both increase BACE1 expression through activation of PKR-eIF2a pathway.^[21]

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 of endosomes in AD.^[22] 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 recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-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.^{[23] [16]} A protein phosphatase-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 induces apoptosis and nuclear translocation, contributing to neuronal death.^[24]

Glycogen synthase kinase 3β (GSK-3β) is responsible for tau phosphorylation and controls several cellular functions including apoptosis. Another study demonstrated that tunicamycin or Aβ treatment can induce PKR activation in human neuroblastoma 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 PKR siRNA. PKR could represent a crucial signaling point relaying stress signals to neuronal pathways by interacting with transcription factor or indirectly controlling GSK3β activation, leading to cellular degeneration in AD.^[25]

Fetal alcohol syndrome

PKR also mediates ethanol-induced protein synthesis inhibition and apoptosis which is linked to fetal alcohol syndrome.^[26]

Interactions

Protein kinase R has been shown to interact with:

• ASK1,^[27]
• DNAJC3,^[28]
• ILF3,^{[29] [30] [31] [32]}
• METAP2,^[33]
• P53,^[34]
• PPP1CA,^[35]
• PRKRA,^{[36] [37]}
• STAT1,^{[38] [39]} and
• TARBP2.^{[40] [41]}

Further reading

• Williams BR . PKR; a sentinel kinase for cellular stress . Oncogene . 18 . 45 . 6112–6120 . November 1999 . 10557102 . 10.1038/sj.onc.1203127 . free .
• García MA, Meurs EF, Esteban M . The dsRNA protein kinase PKR: virus and cell control . Biochimie . 89 . 6–7 . 799–811 . 2007 . 17451862 . 10.1016/j.biochi.2007.03.001 .
• Thomis DC, Doohan JP, Samuel CE . 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 . May 1992 . 1373553 . 10.1016/0042-6822(92)90732-5 .
• McCormack SJ, Thomis DC, Samuel CE . 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 . May 1992 . 1373554 . 10.1016/0042-6822(92)90733-6 .
• Mellor H, Proud CG . A synthetic peptide substrate for initiation factor-2 kinases . Biochemical and Biophysical Research Communications . 178 . 2 . 430–437 . July 1991 . 1677563 . 10.1016/0006-291X(91)90125-Q .
• Meurs E, Chong K, Galabru J, Thomas NS, Kerr IM, Williams BR, Hovanessian AG . Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon . Cell . 62 . 2 . 379–390 . July 1990 . 1695551 . 10.1016/0092-8674(90)90374-N . 20477995 .
• Silverman RH, Sengupta DN . Translational regulation by HIV leader RNA, TAT, and interferon-inducible enzymes . Journal of Experimental Pathology . 5 . 2 . 69–77 . 1991 . 1708818 .
• Roy S, Katze MG, Parkin NT, Edery I, Hovanessian AG, Sonenberg N . Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product . Science . 247 . 4947 . 1216–1219 . March 1990 . 2180064 . 10.1126/science.2180064 . 1990Sci...247.1216R .
• McMillan NA, Chun RF, Siderovski DP, Galabru J, Toone WM, Samuel CE, Mak TW, Hovanessian AG, Jeang KT, Williams BR . 6 . HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR . Virology . 213 . 2 . 413–424 . November 1995 . 7491766 . 10.1006/viro.1995.0014 . free .
• Barber GN, Edelhoff S, Katze MG, Disteche CM . 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 . June 1993 . 7686883 . 10.1006/geno.1993.1262 .
• Squire J, Meurs EF, Chong KL, McMillan NA, Hovanessian AG, Williams BR . Localization of the human interferon-induced, ds-RNA activated p68 kinase gene (PRKR) to chromosome 2p21-p22 . Genomics . 16 . 3 . 768–770 . June 1993 . 7686884 . 10.1006/geno.1993.1263 .
• Prigmore E, Ahmed S, Best A, Kozma R, Manser E, Segal AW, Lim L . A 68-kDa kinase and NADPH oxidase component p67phox are targets for Cdc42Hs and Rac1 in neutrophils . The Journal of Biological Chemistry . 270 . 18 . 10717–10722 . May 1995 . 7738010 . 10.1074/jbc.270.18.10717 . free .
• Barber GN, Wambach M, Wong ML, Dever TE, Hinnebusch AG, Katze MG . Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase . Proceedings of the National Academy of Sciences of the United States of America . 90 . 10 . 4621–4625 . May 1993 . 8099444 . 46564 . 10.1073/pnas.90.10.4621 . free . 1993PNAS...90.4621B .
• Chen ZJ, Parent L, Maniatis T . Site-specific phosphorylation of IkappaBalpha by a novel ubiquitination-dependent protein kinase activity . Cell . 84 . 6 . 853–862 . March 1996 . 8601309 . 10.1016/S0092-8674(00)81064-8 . 112412 . free .
• Kuhen KL, Shen X, Carlisle ER, Richardson AL, Weier HU, Tanaka H, Samuel CE . 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 . August 1996 . 8812437 . 10.1006/geno.1996.0446 .
• Taylor DR, Lee SB, Romano PR, Marshak DR, Hinnebusch AG, Esteban M, Mathews MB . Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR . Molecular and Cellular Biology . 16 . 11 . 6295–6302 . November 1996 . 8887659 . 231632 . 10.1128/mcb.16.11.6295 .
• Kuhen KL, Shen X, Samuel CE . 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 . October 1996 . 8921913 . 10.1016/0378-1119(96)00314-9 .

Notes and References

1. Web site: Entrez Gene: EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2.
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