Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase

Abstract

MAP kinases phosphorylate specific groups of substrate proteins. Here we show that the amino acid sequence FXFP is an evolutionarily conserved docking site that mediates ERK MAP kinase binding to substrates in multiple protein families. FXFP and the D box, a different docking site, form a modular recognition system, as they can function independently or in combination. FXFP is specific for ERK, whereas the D box mediates binding to ERK and JNK MAP kinase, suggesting that the partially overlapping substrate specificities of ERK and JNK result from recognition of shared and unique docking sites. These findings enabled us to predict new ERK substrates and design peptide inhibitors of ERK that functioned in vitro and in vivo. Mitogen-activated protein (MAP) kinases are components of signaling cascades that regulate normal development and pathological processes such as oncogenesis. MAP kinases were identified during biochemical searches for serine/threonine-specific protein kinases stimulated by growth factors in vertebrate cells (for review, see Sturgill and Wu 1991). MAP kinases were also identified in screens for mutations that affect intercellular signaling in yeast, worms, and flies (for review, see Ferrell 1996). Together, these investigations revealed that MAP kinases function in many cell types, are regulated by a diverse group of extracellular stimuli, and mediate a wide variety of cellular responses. MAP kinases can be divided into subfamilies based on specific conserved residues, particularly a TXY motif in the activation loop (Ferrell 1996). The three best-characterized subfamilies in vertebrates are named extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK, also called stress-activated protein kinase), and p38. There are probably several additional vertebrate MAP kinase subfamilies, since Saccharomyces cerevisiae contains six different MAP kinases (Madhani and Fink 1998). Here we use the name MAP kinase to refer to all members of the family, and the names ERK, JNK, and p38 to refer to members of those subfamilies.MAP kinases function in modules composed of three protein kinases (for review, see Marshall 1994). MAP kinase kinase kinases, such as Raf-1, phosphorylate and thereby activate MAP kinase kinases, such as MEK (MAP kinase kinase or ERK kinase). MAP kinase kinases are serine/threonine and tyrosine-specific protein kinases that phosphorylate the TXY motif and thereby activate MAP kinases. In general, MAP kinases in different subfamilies are members of separate modules and are regulated by distinct extracellular stimuli (for review, see Whitmarsh and Davis 1996). For example, ERK is activated strongly by receptor tyrosine kinases (RTK) such as the epidermal growth factor receptor, whereas JNK is activated strongly by stress stimuli such as ultraviolet light. Several of the signaling pathways leading from extracellular stimuli to the activation of a MAP kinase module are well defined, whereas others have yet to be characterized in detail. Whereas the upstream signaling events that r...

MKP5, a new member of the MAP kinase phosphatase family, which selectively dephosphorylates stress-activated kinases

“…The role of this motif in the MAP kinase phosphatases has yet to be established, although it has been suggested that it could serve as an interaction point with cell cycle proteins (Martell et al, 1998). Moreover, the MKP5 protein appears to contain another conserved motif known as the Dbox (Jacobs et al, 1999). Alignment of DNAbinding proteins containing such motifs has resulted in the following consensus:…”

“…This motif has been suggested to function as a docking site that mediates binding to ERK and JNK MAP kinases. The dual-speci®city phosphatase M3/6 (Theodosiou et al, 1996) also contains this consensus whereas MKP1/CL100 and MKP-2 (Keyse and Emslie, 1992; Misra-Press et al, 1995) contain a more highly conserved motif (FXFP) which is speci®c for mediating binding to ERK (Jacobs et al, 1999). Interestingly, in all the above cases, the MAPK docking motifs are located C-terminally to the catalytic domains of the phosphatases as compared to MKP5, where the motif is located Nterminally.…”

“…This enzymatic speci®city is similar to that observed with M3/6 in this type of assay (Muda et al, 1996b and is quite di erent to that of MKP-3. Interestingly, MKP5 and M3/6 are the only members of the family identi®ed thus far, that contain a D-box motif (Jacobs et al, 1999), which could mediate binding to JNK. Further work is required to elucidate the molecular basis of the substrate selectivity of the MAP kinase phosphatases.…”

cRel-TD kinase: a serine/threonine kinase binding in vivo and in vitro c-Rel and phosphorylating its transactivation domain

“…of at least four independent experiments in duplicate PKA-phosphorylation-dependent (Zhong et al, 1998). Importantly, MAP kinases contact their substrates through speci®c docking sites (Jacobs et al, 1999), and compatible DEF and DEJL docking motifs are present both in the c-Rel DNA binding and transactivation domains.…”

Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates

“…A second docking motif (consensus L-X 1-2 -(R/K) [2][3][4][5] ), related to the D-site, is found in MAPK-activated kinases such as RSK1 [50]. Another docking motif for MAP kinases (consensus FXFP) has been named the 'DEF motif' [51]. Fig.…”