Docking Sites on Substrate Proteins Direct Extracellular Signal-regulated Kinase to Phosphorylate Specific Residues

Abstract

Mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK) are important signaling proteins that phosphorylate (S/T)P sites in many different protein substrates. ERK binding to substrate proteins is mediated by docking sites including the FXFP motif and the D-domain. We characterized the sequence of amino acids that can constitute the FXFP motif using peptide and protein substrates. Substitutions of the phenylalanines at positions 1 and 3 had significant effects, indicating that these phenylalanines provide substantial binding affinity, whereas substitutions of the residues at positions 2 and 4 had less effect. The FXFP and D-domain docking sites were analyzed in a variety of positions and arrangements in the proteins ELK-1 and KSR-1. Our results indicate that the FXFP and D-domain docking sites form a flexible, modular system that has two functions. First, the affinity of a substrate for ERK can be regulated by the number, type, position, and arrangement of docking sites. Second, in substrates with multiple potential phosphorylation sites, docking sites can direct phosphorylation of specific (S/T)P residues. In particular, the FQFP motif of ELK-1 is necessary and sufficient to direct phosphorylation of serine 383, whereas the D-domain directs phosphorylation of other (S/T)P sites in ELK-1. The MAP1 kinase superfamily is composed of several subfamilies including ERK, c-Jun N-terminal kinase, and p38 (1, 2). These MAP kinases can be activated by a remarkably diverse set of stimuli that function through a variety of signaling pathways. MAP kinase activation is regulated by two upstream protein kinases; a MAP kinase kinase kinase, such as Raf, phosphorylates and thereby activates a MAP kinase kinase (3, 4). The MAP kinase kinase that regulates ERK is called MEK (MAP kinase kinase or ERK kinase). MEK is a dual specificity protein kinase that phosphorylates a threonine and tyrosine in a TXY motif of ERK, resulting in a significant increase in ERK kinase activity. ERK is inactivated by dual specificity phosphatases that dephosphorylate the TXY motif. Many different stimuli can activate the protein kinase cascade that activates ERK. One extensively characterized activation pathway is initiated by a secreted growth factor, such as epidermal growth factor, that leads to the activation of a receptor tyrosine kinase, Ras, and Raf (5).Signaling pathways that include ERK mediate a remarkably diverse set of responses during the development and homeostasis of organisms such as Caenorhabditis elegans, Drosophila, and vertebrates. The mechanisms that enable highly conserved signaling pathways to elicit cell type-specific responses are being actively investigated, yet remain poorly understood. In principle, any protein in a signaling cascade could function differently in different cell types and, thus, contribute to a specific response. However, ERK is likely to play an important role in generating cell type-specific responses (6). By contrast to Raf and MEK, whose only well documented physiologic...

p38 MAP kinase modulates Smad-dependent changes in human prostate cell adhesion

“…Alternatively, Smads could be direct or indirect targets of p38 MAP kinase phosphorylation, at a site distinct from the TGFbR-mediated phosphorylation/activation, but one that promotes receptor activation of the Smads. Amino-acid sequences that promote direct phosphorylation by p38 MAP kinase have been detected (Fantz et al, 2001;Galanis et al, 2001), but a complete understanding of substrate selection by the kinase remains elusive. Smad2 and Smad3 do possess S/TP motifs, which are phosphorylated by MAP kinases including p38 MAP kinase, but do not contain docking domains similar to the sequences important for p38 MAP kinase phosphorylation of SAP-1 (Galanis et al, 2001).…”

ERK1/2 Achieves Sustained Activation by Stimulating MAPK Phosphatase-1 Degradation via the Ubiquitin-Proteasome Pathway

“…It is of interest to further identify whether ERK1/2 docking on the MKP-1 DEF motif facilitates phosphorylation at the two upstream (S/T)P sites and then affects the stability of MKP-1. Furthermore, studies of Elk-1 and SAP-1 indicate that their DEF motifs and D domains function independently or additively to mediate binding to ERK1/2 for phosphorylation at distinct (S/T)P sites (9,41,42). It is conceivable that association with MKP-1 at the DEF or D domain by ERK1/2 may result in distinct phosphorylation events that control the activity and stability of MKP-1.…”

“…Binding of ERK1/2 to the DEF motifs of several transcriptional factors, including Elk-1, SAP-1, and c-Fos, results in phosphorylation on specific (S/T)P sites positioned upstream from the docking site and subsequent functional activation of these transcriptional factors (41)(42)(43). The MKP-1 Ser 359 and Ser 364 sites located on the C terminus of the DEF motif may not be phosphorylated by ERK1/2 through interaction with the DEF, because this docking/phosphorylation model in Elk-1, SAP-1, and c-Fos has been shown to be position-dependent (41)(42)(43). In fact, MKP-1 contains two (S/T)P sites positioned on the N terminus of the DEF motif.…”

FGF-receptor substrate 2 functions as a molecular sensor integrating external regulatory signals into the FGF pathway

“…The amount of the loaded GST fusion proteins was quantified by amido black staining. [25][26][27], is a shared feature of the activators, effectors, and substrates of ERK2. Although the same sequence was not detected in the aa 248-272 region, the most important components of this domainthe positively charged lysine residues and the hydrophobic leucine residues -were spread throughout the region.…”