Isolation of MEK5 and Differential Expression of Alternatively Spliced Forms

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...

Aberrant expression of extracellular signal-regulated kinase 5 in human prostate cancer

“…We demonstrated a strong, linear correlation between MEK5 and ERK5 protein expression in matched cases (P ¼ 0.0053). This close relationship ties in with current knowledge of ERK5 as the specific substrate for MEK5, and pMEK5 as the only known activating molecule of ERK5 (English et al, 1995;Mody et al, 2003). We have demonstrated a significant correlation between ERK5 cytoplasmic overexpression and Gleason sum score (Po0.0001), an observation not seen for MEK5 expression (Mehta et al, 2003).…”

“…MAP kinase kinases (MEKs/MAPKKs) represent protein kinases upstream of MAPKs, critically controlling cellular proliferation and apoptosis. Mitogen/extracellular signal-regulated kinase kinase-5 (MEK5) is the most recently identified MAPKK and specifically activates ERK5, also known as Big MAP kinase 1 (English et al, 1995;Zhou et al, 1995). The ability of inhibitors of the classical MAPK (ERK1/2) cascade to block ERK5 activation suggested that ERK5 might regulate some cellular functions originally attributed to ERK1/2.…”

Signaling by dual specificity kinases

“…Although, the functional signi®cance of this pathway in relation to other MKK-signaling pathway remains unclear, it is signi®cant to note here that p38MAPK and ERK2 can also phosphorylate and activate MEF2C. In this context, it is interesting to note that MKK5 and ERK5 are highly expressed in cardiac skeletal muscle (Zhou et al, 1995;English et al, 1995) while MEF2 family of transcription factors are mainly expressed in skeletal muscle and brain tissue (Martinet et al, 1994;McDermott et al, 1994). Taken together, these observation may be indicative of the critical role played by MKK5-ERK5 signaling pathway in cardiac physiology (Zhou et al, 1995).…”

“…It is interesting to note that the 23-amino acids encoded by 5' exon in the larger a-isoform is similar to the sequence found in proteins which associate with the actin cytoskeleton. This may be indicative of the isoform-speci®c subcellular localization of MKK5 (English et al, 1995). Interestingly, ERK5 also shows a distinct structural motif consisting of Pro-Ala repeats.…”

“…Two distinct alternatively spliced isoforms of MKK5 have been identi®ed (English et al, 1995). The larger 50-kDa isoform is known as MKK5a whereas the 40-kDa shorter isoform is known as MKK5b.…”