Beata Majchrzak

Activation of the p38 Mitogen-activated Protein Kinase by Type I Interferons

The p38 mitogen-activated protein (Map) kinase plays a critical role in the generation of signals in response to stress stimuli, but its role in interferon (IFN) signaling and its potential regulatory role in the activation of Jak-signal transducer and activator of transcription (Stat) pathway are not known. In the present study, we provide evidence that the p38 Map kinase is rapidly phosphorylated and activated during treatment of cells with Type I interferons (IFNα and IFNβ). Furthermore, the Type I IFN-dependent activation of p38 regulates induction of the catalytic domains of MapKap kinase-2 and MapKap kinase-3, strongly suggesting the existence of an IFNα signaling cascade activated downstream of the p38 kinase. The engagement of this pathway in interferon signaling plays a critical role in interferon-dependent transcriptional regulation, as evidenced by the fact that inhibition of p38 activation results in abrogation of interferon-dependent gene transcription via interferon-stimulated response elements. Interestingly, inhibition of the kinase activity of the p38 blocks IFNα-induced gene transcription without inhibiting DNA binding or tyrosine phosphorylation of Stat proteins, suggesting that the p38 pathway acts in cooperation with the Stat pathway. Thus, the p38 kinase signaling cascade is activated by the Type I interferon receptor and plays a critical role in interferon signaling and interferon-dependent transcriptional regulation.

Protein kinase c-δ (PKC-δ) is activated by Type I interferons and mediates phosphorylation of Stat1 on serine 727

It is well established that engagement of the Type I interferon (IFN) receptor results in activation of JAKs (Janus kinases), which in turn regulate tyrosine phosphorylation of STAT proteins. Subsequently, the IFN-dependent tyrosine-phosphorylated/activated STATs translocate to the nucleus to regulate gene transcription. In addition to tyrosine phosphorylation, phosphorylation of Stat1 on serine 727 is essential for induction of its transcriptional activity, but the IFNα-dependent serine kinase that regulates such phosphorylation remains unknown. In the present study we provide evidence that PKC-δ, a member of the protein kinase C family of proteins, is activated during engagement of the Type I IFN receptor and associates with Stat1. Such an activation of PKC-δ appears to be critical for phosphorylation of Stat1 on serine 727, as inhibition of PKC-δ activation diminishes the IFNα- or IFNβ-dependent serine phosphorylation of Stat1. In addition, treatment of cells with the PKC-δ inhibitor rottlerin or the expression of a dominant-negative PKC-δ mutant results in inhibition of IFNα- and IFNβ-dependent gene transcription via ISRE or GAS elements. Interestingly, PKC-δ inhibition also blocks activation of the p38 MAP kinase, the function of which is required for IFNα-dependent transcriptional regulation, suggesting a dual mechanism by which this kinase participates in the generation of IFNα responses. Altogether, these findings indicate that PKC-δ functions as a serine kinase for Stat1 and an upstream regulator of the p38 MAP kinase and plays an important role in the induction of Type I IFN-biological responses.

Activation of Protein Kinase Cδ by IFN-γ

Engagement of the type II IFN (IFN-γ) receptor results in activation of the Janus kinase-Stat pathway and induction of gene transcription via IFN-γ-activated site (GAS) elements in the promoters of IFN-γ-inducible genes. An important event in IFN-γ-dependent gene transcription is phosphorylation of Stat1 on Ser727, which is regulated by a kinase activated downstream of the phosphatidylinositol 3′-kinase. Here we provide evidence that a member of the protein kinase C (PKC) family of proteins is activated downstream of the phosphatidylinositol 3′-kinase and is engaged in IFN-γ signaling. Our data demonstrate that PKCδ is rapidly phosphorylated during engagement of the type II IFNR and its kinase domain is induced. Subsequently, the activated PKCδ associates with a member of the Stat family of proteins, Stat1, which acts as a substrate for its kinase activity and undergoes phosphorylation on Ser727. Inhibition of PKCδ activity diminishes phosphorylation of Stat1 on Ser727 and IFN-γ-dependent transcriptional regulation via IFN-γ-activated site elements, without affecting the phosphorylation of the protein on Tyr701. Thus, PKCδ is activated during engagement of the IFN-γ receptor and plays an important role in IFN-γ signaling by mediating serine phosphorylation of Stat1 and facilitating transcription of IFN-γ-stimulated genes.

Activation of a CrkL-Stat5 Signaling Complex by Type I Interferons

Type I interferons (IFNα and IFNβ) transduce signals by inducing tyrosine phosphorylation of Jaks and Stats, as well as the CrkL adapter, an SH2/SH3-containing protein which provides a link to downstream pathways that mediate growth inhibition. We report that Stat5 interacts constitutively with the IFN receptor-associated Tyk-2 kinase, and during IFNα stimulation its tyrosine-phosphorylated form acts as a docking site for the SH2 domain of CrkL. CrkL and Stat5 then form a complex that translocates to the nucleus. This IFN-inducible CrkL-Stat5 complex binds in vitro to the TTCTAGGAA palindromic element found in the promoters of a subset of IFN-stimulated genes. Thus, during activation of the Type I IFN receptor, CrkL functions as a nuclear adapter protein and, in association with Stat5, regulates gene transcription through DNA binding.

Activation of Protein Kinase Cδ by All-trans-retinoic Acid

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKCδ, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKCδ. In studies aimed at understanding the functional relevance of PKCδ in the induction of RA responses, we found that pharmacological inhibition of PKCδ (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKCδ associated with retinoic acid receptor-α and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKCδ activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKCδ is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells.

Activation of protein kinase C-delta (PKC-delta) by All-trans-retinoic acid

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKCδ, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKCδ. In studies aimed at understanding the functional relevance of PKCδ in the induction of RA responses, we found that pharmacological inhibition of PKCδ (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKCδ associated with retinoic acid receptor-α and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKCδ activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKCδ is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells.

IFN-γ Activates the C3G/Rap1 Signaling Pathway

IFN-γ transduces signals by activating the IFN-γ receptor-associated Jak-1 and Jak-2 kinases and by inducing tyrosine phosphorylation and activation of the Stat-1 transcriptional activator. We report that IFN-γ activates a distinct signaling cascade involving the c-cbl protooncogene product, CrkL adapter, and small G protein Rap1. During treatment of NB-4 human cells with IFN-γ, c-cbl protooncogene product is rapidly phosphorylated on tyrosine and provides a docking site for the src homology 2 domain of CrkL, which also undergoes IFN-γ-dependent tyrosine phosphorylation. CrkL then regulates activation of the guanine exchange factor C3G, with which it interacts constitutively via its N terminus src homology 3 domain. This results in the IFN-γ-dependent activation of Rap1, a protein known to exhibit tumor suppressor activity and mediate growth inhibitory responses. In a similar manner, Rap1 is also activated in response to treatment of cells with type I IFNs (IFN-α, IFN-β), which also engage CrkL in their signaling pathways. On the other hand, IFN-γ does not induce formation of nuclear CrkL-Stat5 DNA-binding complexes, which are induced by IFN-α and IFN-β, indicating that pathways downstream of CrkL are differentially regulated by different IFN subtypes. Taken altogether, our data demonstrate that, in addition to activating the Stat pathway, IFN-γ activates a distinct signaling cascade that may play an important role in the generation of its growth inhibitory effects on target cells.

Engagement of the CrkL adaptor in interferon α signalling in BCR-ABL-expressing cells

Interferon α (IFNα) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR–ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR–ABL‐expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNα‐signalling in the CML‐derived KT‐1 cell line, which expresses BCR–ABL and is sensitive to the growth inhibitory effects of IFNα. CrkL is constitutively associated with BCR–ABL in these cells and treatment with IFNα had no effect on the BCR–ABL/CrkL interaction. After IFNα stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNα‐dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N‐terminus SH3 domains of CrkL. The resulting CrkL–Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the β‐casein promoter or the promoter of the PML gene, an IFNα‐inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT‐1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR–ABL in CML‐derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti‐proliferative effects of IFNα in CML.

Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha.

We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells. Consistent with this, tyrosine phosphorylation of Stat1 and Stat2 and formation of the IFN-stimulated gene factor 3 (ISGF3) complex occurred to a much higher degree in response to IFN-beta stimulation. These findings demonstrate that differential activation of distinct components of the Jak-Stat pathway by different type I IFN can occur. Furthermore, they strongly suggest that such selective activation accounts for the occurrence of differences in the antiviral properties of distinct type I IFN in certain cell types.

Activation of Mitogen-activated Protein Kinase Kinase (MKK) 3 and MKK6 by Type I Interferons

There is accumulating evidence that the p38 MAP kinase pathway plays important roles in Type I interferon (IFN) signaling, but the mechanisms regulating p38 activation during engagement of the Type I IFN receptor remain to be defined. We sought to identify the events that lead to activation of the p38 MAP kinase in response to Type I IFNs. Our data demonstrate that treatment of sensitive cell lines with IFNα results in activation of both MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6). Such IFN-inducible activation of MKK3 and MKK6 is essential for downstream phosphorylation and activation of the p38 MAP kinase, as shown by studies using mouse embryonic fibroblasts (MEFs) with targeted disruption of the Mkk3 and Mkk6 genes (MKK3–/– MKK6–/–). Similarly, IFN-dependent activation of the downstream effectors of p38, MAPKAPK-2 and MAPKAPK-3, is not detectable in cells lacking Mkk3 and Mkk6, demonstrating that the function of these MAP kinase kinases is required for full activation of the p38 pathway. To define the functional relevance of MKK3/6 engagement in Type I IFN signaling, IFN-inducible gene transcription was evaluated in the MKK3/MKK6 double knock-out cells. IFNα- and IFNβ-dependent transcription via either interferon-stimulated response element or IFNγ activated site elements was defective in MKK3 –/–/MKK6 –/– MEFs in luciferase reporter assays. In addition, IFN-dependent induction of two genes known to be of importance in the generation of IFN responses, Isg15 and Irf-9, was diminished in the absence of Mkk3 and Mkk6. The effects of Mkk3 and Mkk6 on IFN-dependent transcription were unrelated to any effects on the phosphorylation and activation of STAT proteins, indicating the presence of a STAT-independent mechanism. Altogether, our findings demonstrate that MKK3 and MKK6 are rapidly activated during engagement of the Type I IFN receptor and play important roles in Type I IFN signaling and the generation of IFN responses.