Protein Kinase R (PKR) Interacts with and Activates Mitogen-activated Protein Kinase Kinase 6 (MKK6) in Response to Double-stranded RNA Stimulation

Abstract

The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been invoked in different signaling pathways. In cells pre-exposed to the PKR inhibitor 2-aminopurine or in PKR-null cells, the activation of p38 mitogen-activated protein kinase (MAPK) following dsRNA stimulation is attenuated. We found that the p38 MAPK activator MKK6, but not its close relatives MKK3 or MKK4, exhibited an increased affinity for PKR following the exposure of cells to poly(rI:rC), a dsRNA analog. In vitro kinase assays revealed that MKK6 was efficiently phosphorylated by PKR, and this could be inhibited by 2-aminopurine. Expression of kinase-inactive PKR (K296R) in cells inhibited the poly(IC)-induced phosphorylation of MKK3/6 detected by phosphospecific antiserum but did not affect the poly(IC)-induced gel migration retardation of MKK3.This suggests that poly(IC)-mediated in vivo activation of MKK6, but not MKK3, is through PKR. Consistent with this observation, PKR was capable of activating MKK6 as assessed in a coupled kinase assay containing the components of the p38 MAPK pathway. Our results indicate that the interaction of MKK6 and PKR provides a mechanism for regulating p38 MAPK activation in response to dsRNA stimulation. Double-stranded RNAs (dsRNA) 1 formed during virus infection can be potent stimulating agents triggering cellular responses through distinct targets and pathways. DsRNA-dependent interferon synthesis as well as NF-B activation may require the binding of dsRNA with cellular membrane component(s) that includes Toll-like receptor 3 (TLR-3) (1). However, other dsRNA sensor cellular components have also been implicated in the activation of cellular pathways triggered by dsRNA (2-4).The dsRNA-dependent protein PKR is a serine/threonine kinase, the expression of which is induced by interferons. This enzyme is activated in response to dsRNA of viral or synthetic origin, most notably the synthetic polyribonucleotide duplex, poly(rI):poly(rC) (pIC), and also by cytokines and cellular stress signals (5). Once activated, PKR functions as one of the mediators of antiviral and antiproliferative activities of interferons (6 -12). PKR has two double-stranded RNA-binding motifs located in the NH 2 -terminal domain (5, 13), which bind pIC or viral dsRNA intermediates generated during a viral infection and also permit the recruitment of other dsRNA-binding domain-containing proteins (14, 15). The carboxyl-terminal half of PKR harbors the kinase catalytic domain (9). On binding dsRNA, PKR dimerizes and undergoes autophosphorylation at multiple sites (5, 16). The direct antiviral activity of PKR is a consequence of the phosphorylation and activation of its major substrate eukaryotic translation initiation factor eIF2␣, thereby inhibiting protein synthesis and impeding virus multiplication (6,10,12).In addition to playing a role in protein synthesis inhibition, PKR also functions in other biological processes including apoptosis, transcriptional regulation, and cell growth, independent of the phosphorylation of eIF2␣ (5). ...

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

“…16 Moreover, PKR was implicated in the activation of the p38 MAPK in response to dsRNA independently of eIF2a phosphorylation. 17 These findings prompted us to examine a possible role of MAPKs in PKR-mediated cell death by doxorubicin. We observed that doxorubicin treatment resulted in a higher induction of JNK1/2 activity in PKR þ / þ than in PKR À/À MEFs ( Figure 5a, panel a).…”

“…27 Conceivably, it is possible that PKR induces the activity of kinases that act upstream of JNK1/2, based on a previous study showing that activation of p38/MAPK by PKR is mediated by MAPK kinase kinase 6 in cells treated with dsRNA. 17 It is also of interest that PKR physically associates with the apoptosis signal-regulating kinase 1 (ASK1) and promotes ASK1-mediated apoptosis induced by serum deprivation. 28 ASK1 functions upstream of JNK, 29 thus providing a tentative link between PKR and JNK activation.…”

IL-10-Dependent S100A8 Gene Induction in Monocytes/Macrophages by Double-Stranded RNA

“…5C). Moreover, the PKR inhibitor blocks numerous antiviral responses (60) including reduction of MEK6 and p38 MAPK phosphorylation in RAW cells, since the p38 MAPK activator MEK6 has increased affinity for PKR, forming a catalytic complex following exposure of cells to dsRNA (61). Although there are other possible mechanisms, such as inhibition of other signaling molecules, this mechanism could be involved in induction of IL-10 and S100A8, because the p38 pathway was crucial for both (ref.…”

The dsRNA-dependent protein kinase, PKR and cell death

“…125 Aside from NF-kB, PKR has also been shown to phosphorylate mitogen-activated protein kinase 6 (MKK6) and to regulate p38 mitogen-activated protein kinase (MAPK) activation in response to dsRNA stimulation. 126,127 The p38 MAPK cascade regulates a variety of cellular responses to stress, inflammation and cytokines. 59 Activation of these stress-activated kinases has been reported as being defective in cells lacking PKR following exposure to LPS, dsRNA and proinflammatory cytokines.…”