Abstract
Extracellular signal-regulated protein kinase 5 (ERK5) is a recently discovered orphan mitogen-activated protein kinase for which no substrates or strong activators have been described. Two ERK5 chimeras were created as a novel approach to discover its substrates and upstream regulators. One chimeric protein contained the N-terminal domain of the ERK5 catalytic core (subdomains I-IV) and the C-terminal domain of the ERK2 catalytic core (subdomains V-XI). This chimera was highly responsive to stimuli that regulate ERK2 in vitro and in cells. A second chimeric protein consisted of the N-terminal domain of ERK2 (subdomains I-IV) and the C-terminal domain of the ERK5 catalytic core (subdomains V-XI). This chimera was activated in bacteria by coexpression with a constitutively active mutant of MEK1. Using the activated chimera, we identified three in vitro substrates of ERK5. Assaying ERK5 activity in immune complexes with one of these substrates, c-Myc, we determined that the ERK5 catalytic domain is activated by V12 H-Ras and to a lesser extent by phorbol ester but not by constitutively active mutants of Raf-1. Thus, ERK5 is a target of a novel Ras effector pathway that may communicate with c-Myc.
Highlights
Elucidation of signaling pathways from the cell membrane to the nucleus has been furthered by the discovery and analysis of mitogen-activated protein (MAP)1 kinase pathways [1,2,3,4,5,6,7,8,9,10,11,12]
Using an activated form of this chimera, we found that the product of the c-myc proto-oncogene [33, 34] was an Extracellular signal-regulated protein kinase 5 (ERK5) substrate, which facilitated the further analysis of this orphan MAP kinase module
ERK5 contains an extension of approximately 400 residues C-terminal to the conserved MAP kinase core sequence; these residues were truncated from the ERK2/5 chimera so that each chimera could be compared with wild type ERK2
Summary
Elucidation of signaling pathways from the cell membrane to the nucleus has been furthered by the discovery and analysis of mitogen-activated protein (MAP) kinase pathways [1,2,3,4,5,6,7,8,9,10,11,12] Each of these pathways, which have been found in all eukaryotes from unicellular organisms to plants and animals, contains regulatory molecules upstream of a kinase module consisting of a MAP/ERK kinase kinase (MEKK) that phosphorylates and activates a MEK that, in turn, phosphorylates and activates an ERK [13]. To discover novel MAP kinase pathways, we used a PCRbased screen to isolate cDNA clones of previously unknown MEKs, the constituents of the module with the greatest substrate specificity. Because we were not successful in activating MEK5 either in vitro or in vivo, we chose to exploit our understanding of MAP kinase structure to circumvent this problem
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