The Selective Protein Kinase C Inhibitor, Ro-31-8220, Inhibits Mitogen-activated Protein Kinase Phosphatase-1 (MKP-1) Expression, Induces c-Jun Expression, and Activates Jun N-terminal Kinase

Abstract

The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties.These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC. Protein kinase C (PKC)1 is a large and diverse family of protein kinases, whose members are differentially regulated by calcium, phorbol esters, diglycerides, and phosphatidic acid (for a review, see Ref. 1). The various PKC isozymes play important signaling roles in cellular growth, differentiation, and homeostasis. The reported cellular roles of PKC have been identified largely through the use of PKC activators and inhibitors. The bisindolemaleimide class of compounds are staurosporine analogs which by all reports are specific for PKC (2, 3). Two widely used bisindolemaleimides, GF109203X and Ro-31-8220, differ by a single functional group, and although their isozyme specificity varies slightly, they are both potent and selective PKC inhibitors.In addition to cellular responses that may be attributed directly to PKC, PKC signaling pathways likely also contribute to cellular signaling through cross-talk mechanisms with other signaling cascades. Multiple lines of evidence indicate a role for PKC in regulating the ERK and JNK cascades which regulate transcription factors in response to external stimuli. The ERK cascade, a growth factor-stimulated signaling cascade is regulated by the Ras and Raf protooncogenes which activate MEK which in turn activates ERK1 and ERK2 (4, 5). ERK1 and -2 accumulate in the nucleus and phosphorylate and transactivate the transcription factor TCF/Elk-1 (6). A parallel signaling pathway activated by cellular stresses such as uv light and translational inhibitors leads to the activation of MEKK1, SEK, and in turn JNK1 and JNK2 (7, 8). The major role of the JNK signaling pathway is believed to be the...

Essential role of calcium in the regulation of MAP kinase phosphatase-1 expression

“…In murine macrophages expressing an oncogenic DRaf-1: estrogen receptor (DRaf-1-ER) fusion protein, the MKP-1 gene was shown to be induced in response to lipopolysaccharide stimulation but not in response to activation of DRaf-1-ER with estradiol, despite the rapid and prolonged activation of ERK1/ERK2 (Hambleton et al, 1995). Finally, treatment of Rat1 cells with the PKC inhibitor Ro-31-8220 was recently reported to activate the JNK pathway, but to inhibit growth factor-stimulated expression of MKP-1 (Beltman et al, 1996). Taken together, these observations indicate that activation of signalling pathways other than the three known MAP kinase modules is required for full transcription of the MKP-1 gene.…”

Loss of protein kinase C function induces an apoptotic response

“…This view would be consistent with the noted e ects of PKC inhibitors', which in many cases will induce an apoptotic response. However, the use of these kinase inhibitors to assess function, while providing a consistent view of apoptosis induction, is not a su cient criterion for proof given their lack of speci®city (Beltman et al, 1996). Similarly, it has been reported that PKCd is proteolytically activated by an ICE-related protease during apoptosis in U937 cells (Emoto et al, 1996).…”

MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer

“…In order to check if the sensitization of H460MKP1siRNA cells was an event linked to this cell line and also if an alternative method for manipulating MKP1 levels would have the same effect, we used both H-460 and H-23 cell lines, the latter also expressing high basal levels of MKP1 (Vicent et al, 2004). Treatment of both cell lines with the inhibitor of MKP1 expression RO-31-8220 (Beltman et al, 1996) resulted, with a different timing, in a reduction in MKP1 expression to almost undetectable levels ( Figure 6a). H-460 cells treated for 1 h with RO-31-8220 showed an earlier (from 9 to 3 h) and more intense activation of JNK and also p38 than untreated cells (Figure 6b).…”

“…MKP1/CL100 is an immediate-early gene whose expression is regulated by mitogenic, inflammatory and DNA-damage stimuli (Beltman et al, 1996;Sa´nchez-Pe´rez et al, 1998;Li et al, 2001). Although MKP1 levels in normal cells are low, increased levels of MKP1 have been found in human ovarian carcinoma, breast and prostate cancer (Srikanth et al, 1999;Denkert et al, 2002;Wang et al, 2003).…”