Mechanisms and Regulation of Protein Kinase-mediated Signal Transduction in Inflammation, Apoptosis, and Neoplastic Transformation
Signal transduction plays a pivot role in regulating cell functions. Extracellular signals are transmitted into cells via an intracellular signaling network that is composed of multiple signaling pathways, dictating cellular functions, such as growth, differentiation, programmed death (apoptosis) and transformation. Although we have learnt a great deal about the architecture of the intracellular signaling network, our understanding of its biology is limited. The work in my laboratory has been focusing on elucidating molecular mechanisms underlying the biological functions of two important intracellular signaling pathways, c- J un N -terminal protein k inase (JNK) and Ik B k inase (IKK), and understanding the impact of deregulating these signaling pathways on human diseases and cancer.
The JNK and IKK pathways are stimulated by a variety of extracellular signals, including growth factors, cytokines, oncogenes, and environmental stresses and in turn they activate many transcription factors such as c-Jun/AP-1 and NF-kB, as well as non-transcription factor such as members of the Bcl-2 family (Bcl-2, Bcl-XL and BAD). Not surprisingly, it has been implicated that the JNK and IKK pathways play critical roles in many important cellular events, including growth control, differentiation, apoptosis and transformation, as well as in cancer and other human diseases. Thus, understanding the molecular mechanisms that control the functions of the JNK and IKK pathways is important for understanding the biology of the intracellular signal network and should shed light on treatment and prevention of cancer and other human diseases.
My laboratory has long-standing interests in studying the JNK signaling pathway, including the identification and molecularly cloning of the specific JNK-activating kinase JNKK2 (Lu et al., Journal of Biological Chemistry (Communication) 272:24751-24754, 1997) and the creation of the constitutively active JNK1 (JNKK2-JNK1 fusion protein) (Zheng et al., Journal of Biological Chemistry 274:28966-28971, 1999). A new research direction in my laboratory is to study the molecular mechanism underlying the JNK signaling. Recently, we discovered the molecular mechanism underlying the distinct biological functions of, JNK1 and JNK2 (Liu et al., Molecular and Cellular Biology 24:10844-10856, 2004). We found that only JNK1 is significantly activated by many extracellular stimuli that are known to activate JNK, such as TNF, IL-1, UV, TPA and anisomysin, while JNK2 is weakly activated. JNK1 is also responsible for c-Jun activation and apoptosis induced by TNF and UV. By contrast, JNK2 can antagonize activation of JNK1. Our finding resolves a puzzle in the JNK pathway that how two ubiquitously expressed JNK isoforms have different biological functions. In addition, it paves the way for investigators in the field to study the biological functions of JNK in adult animals.
One of the model systems that we have been using to study the biological functions of the JNK signaling pathway is TNF-induced apoptosis. TNF-a is a pleiotrophic cytokine that regulates immune responses, inflammation and apoptosis. TNF-a exerts its biological activities through activating multiple signaling pathways, including JNK, NF-kB and caspase. Activation of caspases is required for apoptotic cell death, whereas IKK activation inhibits apoptosis through the transcription factor NF-kB, whose target genes include anti-apoptotic genes. However, the role of the JNK pathway in TNF-a-induced apoptosis is highly controversial (Lin and Dibling, Aging Cell 1:112-116, 2002; Lin, BioEssays 25:17-24, 2003; Liu and Lin, Cell Research 15:36-42, 2005; Lin, Development Cell , March 2006). We discovered that prolonged, but not transient, JNK activation contributes to TNF-a-induced apoptosis when NF-kB activation is blocked (Tang et al., Nature 414:3313-317, 2001; Tang et al., Molecular and Cellular Biology 22:8571-8579, 2002). Conversion of JNK activation from prolonged to transient suppress TNF-a induced apoptosis in MEFs deficient in IKKbeta or RelA or other cell types (Tang et al., Nature 414:3313-317, 2001; Tang et al., Molecular and Cellular Biology 22:8571-8579, 2002). Furthermore, we found that prolonged JNK activation itself is not sufficient to induce apoptosis (Tang et al., Nature 414:3313-317, 2001). Thus, we proposed a "breaking the brake on apoptosis" model for the role of JNK activation in apoptosis (Lin, BioEssays 25:17-24, 2003). According to this model, JNK activation results in inactivation of apoptotic inhibitors and thereby permits/promotes the apoptotic process. This model can explain why JNK is required for apoptosis but its activation is not sufficient to induce apoptosis.
Another model system we have used to study the biological functions of the JNK signaling pathway is IL-3-mediated survival of hematopoietic cells. The hemotapoietic cytokine IL-3 is essential for survival in many IL-3-dependent hematopoietic progenitor cells. IL-3 exerts its survival effect through activation of several protein kinases, such as protein kinase A (PKA) and another protein kinase Akt. Both PKA and Akt can phosphorylate the proapoptotic Bcl-2 family protein BAD at several serine residues, thereby preventing it to interact and inactivate the antiapoptotic Bcl-2 family protein Bcl-XL. Recently, we discovered the molecular mechanism by which JNK suppresses apoptosis in hemotapoeitic cells (Yu et al., Molecular Cell 13:329-340, 2004). For a long time, the prevailing view in the filed is that JNK acts as a "pro-apoptotic" MAP kinase. However, we found that JNK (JNK1) is activated by IL-3 in IL-3 dependent FL5.12 pre-B cells and inhibition of JNK promotes IL-3-withdrawal induced apoptosis. Activated JNK phosphorylates BAD at Threonine 201 in vitro and in vivo and the phosphorylation decreases the interaction between BAD and Bcl-XL, thereby inhibiting the proapoptotic activity of BAD. Thus, our finding provides a molecular mechanism by which JNK contributes to cell survival in response to hemotapoietic cytokines. In addition, we have also clarified the confusion in the literatures regarding the role of JNK in IL-3-mediated cell survival. It was reported that JNK phosphorylates BAD at serine 128 (Ser128) and thereby promotes cell death. Using a combination of two-dimensional phosphopeptide mapping, phosphoamino acid analysis and site-directed mutagenesis, we demonstrated that JNK does not phosphorylate BAD at Ser128 at all and that elimination of Ser128 phosphorylation has no effects on the proapoptotic activity of BAD in apoptosis induced by UV via JNK or growth factor withdrawal (Zhang et al., Cancer Research 65:8372-8378, 2004).
Another major research topic in my laboratory is to study the signaling mechanisms and biological functions of the IKK-NF-kB signaling pathway. My laboratory has long standing interests in studying the IkB kinase (IKK)-NF-kB signaling pathway, including the identification of MEKK1 as a potential MAP3K for IKK (Nemoto et al., Molecular and Cellular Biology 18:7336-7343, 1998), the elucidation of the IKK-independent molecular mechanism by which pX, the transcription activator of Hepatitis B virus, stimulates NF-kB activity (Purcell et al., American Journal of Physiology 280:G669-677, 2001), the finding that the IKK-NF-kB signaling pathway is required for hypertrophic growth of cardiomyocytes (Purcell et al., Proceedings of the National Academy of Science USA 98:6668-6673, 2001), and more recently the discovery that NF-kB negatively regulates TNF-k-induced JNK activation for cell survival (Tang et al., Nature 414:3313-317, 2001; Tang et al., Molecular and Cellular Biology 22:8571-8579, 2002). Recently, we have been working on the molecular mechanism by which the IKK-NF-kB signaling pathway is "turned-off" in cell death, using TNF-a-induced apoptosis as a model system. Typically, TNF-k is not a killer unless NF-kB activation is impaired. However, TNF-a does induce apoptosis in certain cell types, despite activation of the IKK/NF-kB pathway. We resolved this puzzle by discovering that the IKK/NF-kB pathway is terminated during TNF-a induced apoptosis through the proteolysis of the beta catalytic subunit of the IKK complex (IKKbeta) by caspase 3-related caspases (Tang et al., Molecular Cell 8:1005-1016, 2001). An IKKbeta mutant that is resistant to caspase 3-mediated proteolysis suppressed apoptosis induced by TNF-a as well as Fas ligand. Thus, in addition to activating the proteolytic caspase cascade, cells need to eliminate survival factors such as IKKbeta to carry out TNF-a induced apoptosis. We have generated a caspase-resistant IKKbeta knock-in mouse. The mice are viable and we are in the process to analyze whether the caspase-resistant IKKbeta inhibits apoptosis in vivo and/or sensitizes tumorigenesis.
Another model we have been using to study the biological functions of the NF-kB signaling pathway is to study the regulation of the UV response by NF-kB. The physical stress UV activates both NF-kB and JNK, and induces apoptosis as well. It has been shown by our laboratory and others that JNK1 is essential for UV-induced apoptosis (Liu et al., Molecular and Cellular Biology 24:10844-10856, 2004). Although NF-kB is anti-apoptotic in most cases, NF-kB promotes UV-induced apoptosis. However, the underlying mechanism is incompletely understood. Most recently, we discovered that RelA/NF-kB is essential for rapid and robust activation of JNK1 by UV (Liu et al., Molecular Cell 21:467-480 , 2006). In resting cells, the preexisting nuclear RelA, which is a major transactivating subunit of NF-kB, has already been recruited to the promoter of PKCdelta and is required for its expression. In response to UV-irradiation, PKCdelta phosphorylates JNK1 at Ser129 and thereby ensures its phosphorylation by its upstream kinases (JNKK1 and JNKK2) in the phosphorylation-activation loop at Thr183 and Tyr185. The rapid and robust activation of JNK1 by UV is required for UV-induced apoptosis as it is involved in the onset of UV killing. Thus, our discovery revealed a novel positive regulation of JNK by NF-kB and provided a novel molecular mechanism by which NF-kB promotes UV-induced apoptosis. Furthermore, our discovery provides an interesting paradigm in the field of signal transduction. Previously, we discovered that NF-kB negatively regulates TNF-induced JNK activation for cell survival (Tang et al., Nature 414:3313-317, 2001). Our current discovery shows that NF-kB positively regulates UV-induced JNK activation for cell death (Liu et al., Molecular Cell 21:467-480 , 2006). Thus, the crosstalk between two (or multiple) given signaling pathways can be different in response to distinct extracellular stimuli, thereby providing the specificity of signal transduction.
Another new model we have been using to study the biological functions of the NF-kB signaling pathway is to investigate the crosstalk between NF-kB and the second messenger cAMP. For more than two decades, it has been known that cAMP is able to promote cell death in certain types of cells, such as CD4+CD8+ (double positive) T cells in thymus. Yet the underlying molecular mechanism is incompletely understood. Recently, we discovered that cAMP inhibits TNF-induced NF-kB activation via the PKA-CREB-DLC signaling pathway (Zhang et al., Molecular and Cellular Biology, 26:1223-1234, 2006). Once activated by cAMP, PKA translocates into the nucleus where it phosphorylates and activates the transcription factor CREB. This leads to upregulation of DLC, which is encoded by the CREB target gene dlc . We found DLC is able to interrupt the interaction between the MAP kinase p38 and its upstream kinases MKK3/6, thereby inhibiting activation of p38, the latter is involved in the activation of NF-kB in the nucleus. Since NF-kB is essential for suppressing TNF killing, inhibition of p38 by the cAMP-PKA-CREB-DLC results in suppression of NF-kB activation and promotion of TNF-induced apoptosis. Thus, our discovery provides a molecular mechanism by which cAMP promotes apoptosis.
In addition to investigating the molecular mechanisms and biological functions of signal transduction, we have studied how deregulation of signal transduction contribute to diseases and cancer, using cardiac hypertrophy, breast and prostate cancer as model systems (Nemoto et al., Molecular and Cellular Biology, 18:3518-3526, 1998; Purcell et al., Proceedings of the National Academy of Science USA 98:6668-6673, 2001; Tang et al., Molecular and Cellular Biology 22:8571-8579, 2002). Our work may identify novel targets for prevention and treatment of human diseases and cancer.