No abstract

The prototype mitogen-activated protein (MAP) kinase module is a three-kinase cascade consisting of the MAP kinase, extracellular signal-regulated protein kinase (ERK) 1 or ERK2, the MAP/ERK kinase (MEK) MEK1 or MEK2, and the MEK kinase, Raf-1 or B-Raf. This and other MAP kinase modules are thought to be critical signal transducers in major cellular events including proliferation, differentiation, and stress responses. To identify novel mammalian MAP kinase modules, polymerase chain reaction was used to isolate a new MEK family member, MEK5, from the rat. MEK5 is more closely related to MEK1 and MEK2 than to the other known mammalian MEKs, MKK3 and MKK4. MEK5 is thought to lie in an uncharacterized MAP kinase pathway, because MEK5 does not phosphorylate the ERK/MAP kinase family members ERK1, ERK2, ERK3, JNK/SAPK, or p38/HOG1, nor will Raf-1, c-Mos, or MEKK1 highly phosphorylate it. Alternative splicing results in a 50-kDa ␣ and a 40-kDa ␤ isoform of MEK5. MEK5␤ is ubiquitously distributed and primarily cytosolic. MEK5␣ is expressed most highly in liver and brain and is particulate. The 23 amino acids encoded by the 5 exon in the larger ␣ isoform are similar to a sequence found in certain proteins believed to associate with the actin cytoskeleton; this alternatively spliced modular domain may lead to the differential subcellular localization of MEK5␣.A common element in many eukaryotic regulatory pathways is a three-kinase cascade, known as a MAP 1 kinase module. A module consists of three protein kinases that act sequentially within a pathway: a MAP kinase kinase kinase or MEKK (a MEK activator), a MAP kinase/ERK kinase or MEK (a MAP kinase activator), and a MAP kinase or ERK (extracellular signal-regulated protein kinase) homolog. First recognized in yeast (1), several MAP kinase modules have now been identified in mammalian systems (2, 3). This kind of three-kinase regulatory cascade conveys information to target effectors, coordinates incoming information from parallel signaling pathways, confers a vast potential for amplification and specificity, and incorporates multiple inactivation mechanisms. The first and best studied is the MAP kinase pathway, made up of Raf-1 or B-Raf, MEK1 or MEK2, and ERK1 or ERK2 (4).The closely related MAP kinases, ERK1 and ERK2 (5-8), are ubiquitous signal transducers. They are activated by diverse extracellular stimuli and by proto-oncogene products that induce proliferation or enhance differentiation (7). In addition to ERK1 and ERK2, other related mammalian enzymes have been detected including: several ERK3 isoforms (7, 9, 10), ERK4 (11), Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs) (12, 13), p38/HOG1 (14 -16), and p57 MAP kinases (17). The presence of at least six MAP kinases in yeast (18) coupled with Southern analysis of rat DNA (7) suggest that there are numerous MAP kinase relatives and a corresponding number of MAPK kinase modules in mammals.The two dual specificity MAP kinase kinases, , are the only known enzymes capable of phosphorylating and ac...

We showed previously that ERK1/2 were activated by glucose and amino acids in pancreatic ␤ cells. Here we examine and compare signaling events that are necessary for ERK1/2 activation by glucose and other stimuli in ␤ cells. We find that agents that interrupt Ca 2؉ signaling by a variety of mechanisms interfere with glucose-and glucagon-like peptide (GLP-1)-stimulated ERK1/2 activity. In particular, calmodulin antagonists, FK506, and cyclosporin, immunosuppressants that inhibit the calcium-dependent phosphatase calcineurin, suppress ERK1/2 activation by both glucose and GLP-1. Ca 2؉ signaling from intracellular stores is also essential for ERK1/2 activation, because thapsigargin blocks ERK1/2 activation by glucose or GLP-1. The glucosesensitive mechanism is distinct from that used by phorbol ester or insulin to stimulate ERK1/2 but shares common features with that used by GLP-1.

The small GTP-binding protein Ras appears to be required for transformation and differentiation induced by tyrosine kinases. The Ras requirement may be limited to a few tyrosine kinase-regulated signaling pathways or may be universal for all tyrosine kinase actions. Because both Ras and the microtubule-associated protein 2 kinases ERKi and ERK2 have been implicated in events that lead to neurite outgrowth, we explored the possibility that Ras and ERKs may lie on the same signaling pathway. Uzing PC-12 rat adrenal pheochromocytoma cell lines that contain a dominant inhibitory Ras mutant (S17N-RasH), we found that Ras was required for stimulation of the ERK cascade by nerve growth factor but apparently not by the heterotrimeric G protein activator AIF4 . Within this cascade, Ras appears to be upstream of an ERK activator, raising the intrigung psibility that Ras may directly regulate a serine/threonine protein kinase.

The coding sequence of rat MEK kinase 1 (MEKK1) has been determined from multiple, independent cDNA clones. The cDNA is full-length based on the presence of stop codons in all three reading frames of the 5' untranslated region. Probes from the 5' and the 3' coding sequences both hybridize to a 7-kb mRNA. The open reading frame is 4.5 kb and predicts a protein with molecular mass of 161,225 Da, which is twice the size of the previously published MEKK1 sequence and reveals 801 amino acids of novel coding sequence. The novel sequence contains two putative pH domains, two proline-rich regions, and a cysteine-rich region. Antisera to peptides derived from this new sequence recognize an endogenous protein in human and rodent cells of 195 kDa, consistent with the size of the expressed rat MEKK1 clone. Endogenous and recombinant rat MEKK1 are enriched in membranes; little of either is found in soluble fractions. Expression of recombinant rat MEKK1 leads to activation of three mitogen-activated protein kinase modules in the order c-Jun N-terminal kinase/stress-activated protein kinase > p38 mitogen-activated protein kinase = extracellular signalregulated kinase 2.