T he mitogen-activated protein kinase (MAPK) family consists of a group of kinases responsive to a variety of environmental stimuli. MAPKs can be subdivided into three groups: extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38MAPK (1-8). Although ERK has been shown to be activated primarily by proliferative signals, JNK and p38MAPK are activated by genotoxic as well as cytotoxic stress signals (7-10). The structural organization of these kinases into specific signaling modules appears to be facilitated by scaffolding proteins such as STE5 in yeast (11)(12)(13)(14) and JNK͞stress-activated protein kinase-associated protein (JSAP) and JNK interacting proteins 1-3 in mammalian cells (15)(16)(17)(18)(19)(20). These scaffolding proteins tether different MAPK kinase kinases (MEKKs), MAPK kinases (MKKs), and MAPKs into close proximity so that the successive phosphorylation events can occur efficiently, thus conferring specificity to a particular combination of kinases for activation. Although it is well documented that these phosphorylation cascades lead to the activation of transcription factors such as Fos, Jun, and Myc, the precise mechanisms through which the specific transcription factors are recruited is not known (1, 4). In our search for proteins that associate with transcription factors such as Max and Myc, we identified a scaffolding protein, which we termed JLP for JNK-associated leucine zipper protein. Here we show that JLP brings together Max and c-Myc along with JNK and p38MAPK, as well as their upstream kinases MKK4 and MEKK3. Thus, JLP defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways. Materials and MethodsCloning of JLP. Human Max was expressed as bacterial recombinant protein, which was 32 P-labeled in vitro with heart muscle kinase (Sigma) by using [␥-32 P]ATP. The Max probe was added in the hybridization buffer Hyb75 (20 mM Hepes-KOH, pH 7.7͞75 mM KCl͞0.1 mM EDTA͞2.5 mM MgCl 2 ͞0.05% Nonidet P-40͞1% nonfat milk͞10 mM DTT) and used to screen the gt11 expression library derived from 32Dcl3 cells (21) as described (22). Four overlapping cDNAs were used to generate a cDNA sequence encoding for the full-length protein of JLP.S Tagging and Mutation of JLP. PCR was used to generate a fragment of JLP sequence (3,250-4,083 bp) whose 3Ј end contained the coding sequence of S tag (KETAAAKFERQH-MDS) followed by a stop codon. Substitution of all leucine residues [amino acids 117, 124, 131, 145, 152, and 159 in leucine zipper I (LZI) and amino acids 413 and 420 in leucine zipper II (LZII)] of JLP with alanine residues was carried out by sitedirected mutagenesis and fusion PCR by using M2 cDNA as the template as described (23). All mutations and deletion constructs were verified by sequence analysis. JLP-S 3Ј deletion mutants were created by digestion of the WT JLP-S cDNA EcoRI͞MluI fragment with SmlI, MslI, PvuII, BssSI, SpeI, and Bsu36I. JLP-S domain constructs were generate...

No abstract

The protooncogene c-myb encodes a nuclear transcription factor that binds to DNA in a sequence-specific manner and transactivates transcription of several viral and cellular genes. The expression of c-myb is induced in mitogenstimulated peripheral blood lymphocytes and is constitutively expressed in several CD4' T-cell

During hexamethylene bisactamide (HMBA)-induced dierentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while cmyb and several other nuclear proto-oncogenes are down-regulated. Dierentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell dierentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of cmyb. The cAMP-induced NF-kB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kB. We conclude that inhibition of MEL cell dierentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kB p50/RelB heterodimers.