Phosphorylation of MAP Kinases by MAP/ERK Involves Multiple Regions of MAP Kinases

Abstract

Mitogen-activated protein (MAP) kinases are activated with great specificity by MAP/ERK kinases (MEKs). The basis for the specific activation is not understood. In this study chimeras composed of two MAP kinases, extracellular signal-regulated protein kinase 2 and p38, were assayed in vitro for phosphorylation and activation by different MEK isoforms to probe the requirements for productive interaction of MAP kinases with MEKs. Experimental results and modeling support the conclusion that the specificity of MEK/MAP kinase phosphorylation results from multiple contacts, including surfaces in both the N-and C-terminal domains. Mitogen-activated protein (MAP)1 kinase or extracellular signal-regulated protein kinase (ERK) cascades are present in all eukaryotes and are utilized in almost all signal transduction pathways originating from receptors at the cell surface (1, 2). A plethora of different stimuli, including growth factors, cytokines, heat shock, and ultraviolet light, can initiate signaling through these cascades. Each cascade consists of a three kinase module: a MAP kinase, a MAP kinase/ERK kinase (MEK) that activates the MAP kinase, and a MEK kinase (MEKK) that activates the MEK (3). The MAP kinase in each cascade preferentially phosphorylates substrates with a serine or threonine followed by a proline. There are three mammalian MAP kinase modules that have been extensively studied. These include the ERK1/2 module, the c-Jun N-terminal protein kinase/stressactivated protein kinase module, and the p38 module. ERK3, ERK4, and ERK5 and other p38 isoforms have also been identified, but the cascades leading to activation of these kinases are not well characterized (4 -11).Like other protein kinases, the MAP kinases are folded into two domains (12). The smaller N-terminal domain is composed mostly of ␤ strands, whereas the C-terminal domain is made up of ␣ helices. ATP binds between the two domains, and protein substrate is believed to bind on the surface of the C-terminal domain. Alignment of the amino acid sequences of many protein kinases reveals a common core catalytic domain of 250 -300 residues encoding the two-domain structure (13). Protein kinases possess 12 conserved stretches of amino acids within their catalytic domains known as subdomains (13-15). These conserved elements as well as unique structures contribute to catalysis by and regulation of protein kinases. The functions of several of the conserved and unique structural motifs are known. A glycine-rich loop in the N-terminal domain, termed the phosphate anchor ribbon, has a role in binding ATP. Also in the N-terminal domain, subdomain III encodes the C helix, which contains an invariant Glu involved in binding MgATP (16,17). This helix is important for maintaining an open domain conformation in unphosphorylated ERK2 (18), aligning catalytic residues in Src (19) and Cdk2 (20), and binding to the cyclin regulatory subunit in Cdk2 (21). The activation loop, known as the phosphorylation lip in MAP kinases, is a poorly conserved element in the C-termi...

PEA-15 Binding to ERK1/2 MAPKs Is Required for Its Modulation of Integrin Activation

“…The p38/ERK1 chimeric constructs have been described (12). The p38/ERK2 constructs were gifts from Dr. Melanie Cobb (13). The HA-ERK1-CAAX-pEF and HA-ERK1-SAAX-pEF constructs were as described (15).…”

“…To map the regions of ERK1/2 involved in PEA-15 binding, we employed a number of existing ERK/p38 chimeras (Fig. 3A) used previously to identify domains important for interacting with upstream kinases, target substrates, and phosphatases (12,13,21). The three-dimensional structures of ERK1/2 and p38 are similar (22), and the chimeras contain selected intact structural subdomains from each enzyme.…”

“…Sequence derived from ERK1/2 and p38 are represented by white and gray boxes, respectively. The chimeras are named according to the subdomains that were exchanged (12,13). Chimera PIVE contains p38 sequence constituting the N terminus up to and including subdomain IV with the remaining sequence from ERK2.…”

“…Single point mutations in either the DED or tail of PEA-15, which inhibit interaction with ERK1/2, resulted in a concomitant loss of the ability of PEA-15 to reverse suppression of integrin activation. Furthermore, we used a series of ERK/ p38 chimeras (12,13) in combination with site-directed mutagenesis to identify ERK1/2 residues required for PEA-15 binding. Point mutation of residues preceding the ␣G helix or within the MAPK insert blocked the capacity of ERK2 to bind PEA-15 but did not adversely effect ERK2 phosphorylation.…”

The Death Effector Domain Protein PEA-15 Prevents Nuclear Entry of ERK2 by Inhibiting Required Interactions

“…MEK1 contacts a significant portion of the ERK2 surface (9). Among well defined binding contacts, the MAP kinase insert is the site that is required for productive interaction between ERK2 and MEK1 that mediates ERK2 phosphorylation by MEK1 (11).…”