The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.

The JNK protein kinase is a member of the MAP kinase group that is activated in response to dual phosphorylation on threonine and tyrosine. Ten JNK isoforms were identified in human brain by molecular cloning. These protein kinases correspond to alternatively spliced isoforms derived from the JNK1, JNK2 and JNK3 genes. The protein kinase activity of these JNK isoforms was measured using the transcription factors ATF2, Elk-1 and members of the Jun family as substrates. Treatment of cells with interleukin-1 (IL-1) caused activation of the JNK isoforms. This activation was blocked by expression of the MAP kinase phosphatase MKP-1. Comparison of the binding activity of the JNK isoforms demonstrated that the JNK proteins differ in their interaction with ATF2, Elk-1 and Jun transcription factors. Individual members of the JNK group may therefore selectively target specific transcription factors in vivo.

Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.

The product of the c-myc gene (c-Myc) is a sequence-specific DNA-binding protein that has previously been demonstrated to be required for cell cycle progression. Here we report that the c-Myc DNA binding site confers cell cycle regulation to a reporter gene in Chinese hamster ovary cells. The observed transactivation was biphasic with a small increase in G1 and a marked increase during the S-to-G2/M transition of the cell cycle. This cell cycle regulation of transactivation potential is accounted for, in part, by regulatory phosphorylation of the c-Myc transactivation domain. Together, these data demonstrate that c-Myc may have an important role in the progression of cells through both the G1 and G2 phases of the cell cycle.

The product of the human c-myc protooncogene (Myc) is a sequence-specific DNA binding protein. Here, we demonstrate that the placement of the specific Myc DNA binding site CACGTG upstream of a luciferase reporter gene conferred Myc-stimulated expression that was inhibited by the overexpression of the basic-helix-loop-helix/leucine zipper protein Max. It was observed that Myc was phosphorylated in vivo within the NH2-terminal domain at Thr-58 and Ser-62. Replacement of these phosphorylation sites with Ala residues caused a marked decrease in Myc-stimulated reporter gene expression. In contrast, the replacement of Thr-58 or Ser-62 with an acidic residue (Glu) caused only a small inhibition of transactivation. Together, these data demonstrate that the NH2-terminal phosphorylation sites Thr-58 and Ser-62 are required for high levels of transactivation of gene expression by Myc.

The nucleus is an important target of signal transduction by growth factor receptors that stimulate mitogen-activated protein (MAP) kinases. We tested the hypothesis that MAP kinases have a signaling role within the nucleus by examining the effect of the expression of a human MAP kinase isoform (p41mapk) in tissue culture cells. The expressed p41mapk was found to be localized in both the cytoplasmic and nuclear compartments of the cells. Significantly, the expression of p41mapk caused an increase in the phosphorylation of a nuclear substrate: Ser62 of c-Myc. Phosphorylation at Ser62 stimulated the activity of the NH2-terminal transactivation domain of c-Myc. Thus, p41mapk causes the phosphorylation and regulation of a physiologically significant nuclear target of signal transduction. These data establish that at least one MAP kinase isoform has a nuclear role during signal transduction.

The c-myc gene encodes a sequence-specific DNA-binding protein (c-Myc) that forms leucine zipper complexes and can act as a transcription factor. Growth factor stimulation of cells causes the phosphorylation of the c-Myc transcriptional activation domain at Ser62 within a proline-rich region that is highly conserved among members of the Myc family (Alvarez, E., Northwood, I.C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). This phosphorylation site is a substrate for growth factor-regulated MAP kinases and for the cell cycle-dependent protein kinase p34cdc2. We report that serum treatment of cells results in a marked increase in the transactivation of gene expression mediated by the c-Myc transcriptional activation domain. A point mutation at the site of growth factor-stimulated phosphorylation (Ser62) decreases the serum induction of transactivation. These data indicate that the c-Myc transcriptional activation domain may be a direct target of signal transduction pathways.