Marie A. Bogoyevitch

Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases

SUMMARY The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin.

The pattern of protein kinase C (PKC) isotype expression in whole extracts of dispersed, freshly isolated adult rat ventricular myocytes and adult rat heart ventricle was examined by immunoblot analysis using antisera specific for PKC-alpha, -beta 1, -gamma, -delta, -epsilon, -zeta, or -eta isotypes. This analysis revealed significant levels of expression of the Ca(2+)-independent isotype PKC-epsilon, which was detected as band of 97-kd molecular mass. PKC-zeta was detected principally as a 66-kd band that probably represented a proteolytic product of the holoenzyme. PKC-eta was detected only in whole ventricle as a doublet at 75 and 81 kd and was therefore probably present in nonmyocytic cells. PKC-alpha, -beta 1, -gamma, and -delta could not be detected. Because of our inability to detect PKC-alpha, -beta 1, -gamma, and -delta in whole extracts, PKC isotypes were partially purified from whole heart by DEAE Sepharose chromatography. PKC-alpha, -beta 1, -gamma, and -delta could still not be detected in the appropriate fractions. All PKC isotypes were detectable in appropriate positive control extracts (brain or certain cultured cell lines). In unstimulated isolated cardiomyocytes, the majority (80-95%) of the PKC-epsilon immunoreactivity was present in the soluble fraction of the extract. On exposure of the cardiomyocytes to 1 microM phorbol 12-myristate 13-acetate (PMA), PKC-epsilon undergoes a rapid (< 30 seconds), sustained (at least 60 minutes), and virtually complete association with the Triton X-100-soluble membrane fraction. There was an associated loss of PKC-epsilon from the soluble fraction. The EC50 for PMA of the translocation event was 15-37 nM. Exposure of cardiomyocytes to 1 microM 4 beta-phorbol 12,13-didecanoate or 1 microM phorbol 12,13-dibutyrate also resulted in translocation of PKC-epsilon to the membrane fraction, whereas exposure to 1 microM 4 alpha-phorbol 12,13-didecanoate was without effect. PKC-epsilon also translocated on exposure of cardiomyocytes to 50 microM epinephrine or 100 nM endothelin-1. However, in both cases, the extent of translocation was significantly less than that after exposure to PMA. We conclude that interventions that lead to hypertrophy of cardiomyocytes (phorbol esters, epinephrine, and endothelin-1) activate PKC-epsilon.

The Role of Stem Cells in Skeletal and Cardiac Muscle Repair

In postnatal muscle, skeletal muscle precursors (myoblasts) can be derived from satellite cells (reserve cells located on the surface of mature myofibers) or from cells lying beyond the myofiber, e.g., interstitial connective tissue or bone marrow. Both of these classes of cells may have stem cell properties. In addition, the heretical idea that post-mitotic myonuclei lying within mature myofibers might be able to re-form myoblasts or stem cells is examined and related to recent observations for similar post-mitotic cardiomyocytes. In adult hearts (which previously were not considered capable of repair), the role of replicating endogenous cardiomyocytes and the recruitment of other (stem) cells into cardiomyocytes for new cardiac muscle formation has recently attracted much attention. The relative contribution of these various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are the focus of this review. We concluded that, although many endogenous cell types can be converted to skeletal muscle, the contribution of non-myogenic cells to the formation of new postnatal skeletal muscle in vivo appears to be negligible. Whether the recruitment of such cells to the myogenic lineage can be significantly enhanced by specific inducers and the appropriate microenvironment is a current topic of intense interest. However, dermal fibroblasts appear promising as a realistic alternative source of exogenous myoblasts for transplantation purposes. For heart muscle, experiments showing the participation of bone marrow-derived stem cells and endothelial cells in the repair of damaged cardiac muscle are encouraging.

The c-Jun N-terminal protein kinase family of mitogen-activated protein kinases (JNK MAPKs).

The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPKs) are an evolutionarily-conserved family of serine/threonine protein kinases. First identified in 1990 when intraperitoneal injection of the protein synthesis inhibitor cycloheximide activated a 54 kDa protein kinase, the JNK MAPKs have now taken on a prominent role in signal transduction. This research has revealed a number of levels of complexity. Alternative gene splicing is now recognised to result in ten different JNK MAPK isoforms of 46-55 kDa, and these isoforms differ in their substrate affinities. Furthermore, although originally classified as stress-activated protein kinases (SAPKs), or SAPKs, the JNK MAPKs are also critical mediators of signal transduction in response to stimulation by cytokines and some growth factors. JNK MAPKs have been shown to be critical mediators in dorsal closure in developing Drosophila embryos, and targeted knockout of murine JNK MAPKs has suggested a critical involvement of these kinases in mammalian embryonic development. Recent work has also highlighted their importance in programmed cell death. Thus, the JNK MAPKs may provide a critical target for regulation in both normal and diseased states.

Identification of the critical features of a small peptide inhibitor of JNK activity.

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). Although progress in evaluating the functions of other MAPKs has been facilitated by the characterization of specific inhibitors, no JNK-directed inhibitor is commercially available. We have identified a 21-amino acid peptide inhibitor of activated JNKs, based on amino acids 143–163 of the JNK-binding domain (JBD) of the JNK scaffolding protein, JNK-interacting protein-1 (JIP-1). This peptide, I-JIP (Inhibitor of JNK-based on JIP-1), inhibited JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 up to 90%. A truncated I-JIP (TI-JIP), the C-terminal 11 amino acids of I-JIP, directly interacted with recombinant JNKs but not its substrates as shown by surface plasmon resonance analysis. Scanning alanine replacement within truncated I-JIP identified 4 residues (Arg-156, Pro-157, Leu-160, or Leu-162) as independently critical for inhibition. JBD peptide sequences from JIP-2 and JIP-3 shared these critical residues and accordingly were effective JNK inhibitors. In contrast, peptides based on the JBDs of ATF2 and c-Jun inhibited JNK activity by <40%, which agreed with their lack of homology to the critical Arg-156 and Pro-157. These studies thus define a small peptide inhibitor sequence of JNKs based on the JIP proteins.

c-Jun N-terminal kinase (JNK) signaling: recent advances and challenges.

c-Jun N-terminal kinases (JNKs), first characterized as stress-activated members of the mitogen-activated protein kinase (MAPK) family, have become a focus of inhibitor screening strategies following studies that have shown their critical roles in the development of a number of diseases, such as diabetes, neurodegeneration and liver disease. We discuss recent advances in the discovery and development of ATP-competitive and ATP-noncompetitive JNK inhibitors. Because understanding the modes of actions of these inhibitors and improving their properties will rely on a better understanding of JNK structure, JNK catalytic mechanisms and substrates, recent advances in these areas of JNK biochemistry are also considered. In addition, the use of JNK gene knockout animals is continuing to reveal in vivo functions for these kinases, with tissue-specific roles now being dissected with tissue-specific knockouts. These latest advances highlight the many challenges now faced, particularly in the directed targeting of the JNK isoforms in specific tissues.

Hypertrophic Agonists Stimulate the Activities of the Protein Kinases c-Raf and A-Raf in Cultured Ventricular Myocytes

We detected expression of two Raf isoforms, c-Raf and A-Raf, in neonatal rat heart. Both isoforms phosphorylated, activated, and formed complexes with mitogen-activated protein kinase kinase 1 in vitro. However, these isoforms were differentially activated by hypertrophic stimuli such as peptide growth factors, endothelin-1 (ET1), or 12-O-tetradecanoylphorbol-13-acetate (TPA) that activate the mitogen-activated protein kinase cascade. Exposure of cultured ventricular myocytes to acidic fibroblast growth factor activated c-Raf but not A-Raf. In contrast, TPA produced a sustained activation of A-Raf and only transiently activated c-Raf. ET1 transiently activated both isoforms. TPA and ET1 were the most potent activators of c-Raf and A-Raf. Both utilized protein kinase C-dependent pathways, but stimulation by ET1 was also partially sensitive to pertussis toxin pretreatment. c-Raf was inhibited by activation of cAMP-dependent protein kinase although A-Raf was less affected. Fetal calf serum, phenylephrine, and carbachol were less potent activators of c-Raf and A-Raf. These results demonstrate that A-Raf and c-Raf are differentially regulated and that A-Raf may be an important mediator of mitogen-activated protein kinase cascade activation when cAMP is elevated.

Intracellular signalling through protein kinases in the heart

Protein kinases play important roles in intracellular signalling pathways in probably all cells. In the heart, they are involved in the regulation of ion handling, contractility, fuel metabolism and growth. In this review, we discuss the consequences of activation of protein kinases known to be expressed in the heart. We concentrate principally on the following: cyclic AMP-dependent protein kinase, protein kinase C, mitogen-activated protein kinase, Ca2+/calmodulin-dependent protein kinases and pyruvate dehydrogenase kinase.

Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes

ET‐1 stimulated MBP kinase activity in cultured cardiomyocytes. Maximal activation (3.5‐fold) was at 5 min. EC50 was 0.2 nM. PMA or PE also increased MBP kinase (4‐ or 2.5‐fold, respectively). Pre‐treatment with PMA down‐regulated the subsequent response to ET‐1 or PMA. ET‐1‐ or PMA‐stimulated MBP kinase was resolved into 2 major (peaks II and IV) and 2 minor peaks by FPLC on Mono Q. Peaks II and IV were inactivated by either LAR or PP2A. Renatured MBP kinase activities following SDS‐PAGE in MBP‐containing gels and immunoblot analysis showed that peak II was a p42 MAP kinase and peak IV was a p44 MAP kinase.

Inhibitors of c-Jun N-terminal kinases: JuNK no more?

Abstract The c-Jun N-terminal kinases (JNKs) have been the subject of intense interest since their discovery in the early 1990s. Major research programs have been directed to the screening and/or design of JNK-selective inhibitors and testing their potential as drugs. We begin this review by considering the first commercially-available JNK ATP-competitive inhibitor, SP600125. We focus on recent studies that have evaluated the actions of SP600125 in lung, brain, kidney and liver following exposure to a range of stress insults including ischemia/reperfusion. In many but not all cases, SP600125 administration has proved beneficial. JNK activation can also follow infection, and we next consider recent examples that demonstrate the benefits of SP600125 administration in viral infection. Additional ATP-competitive JNK inhibitors have now been described following high throughput screening of small molecule libraries, but information on their use in biological systems remains limited and thus these inhibitors will require further evaluation. Peptide substrate-competitive ATP-non-competitive inhibitors of JNK have also now been described, and we discuss the recent advances in the use of JNK inhibitory peptides in the treatment of neuronal death, diabetes and viral infection. We conclude by raising a number of questions that should be considered in the quest for JNK-specific inhibitors.