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MAP Kinase Pathways Research Tools
T
he mitogen-activated protein (MAP) kinases are a group of protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus.1,2 In combination with several other signaling pathways, they can differentially alter the phosphorylation status of transcription factors. The controlled regulation of MAP kinase cascades is involved in cell proliferation and differentiation, whereas an unregulated activation can result in oncogenesis.3-5
Three major types of MAP kinase cascades have been reported in mammalian cells that respond synergistically to different upstream signals. The most widely studied cascade is that of ERK1/ERK2 MAP kinases. A general activation scheme involves the activation of receptor tyrosine kinases by growth factors, such as EGF, which provides the binding site for the adapter protein Grb2 which in turn localizes Sos to the plasma membrane. Sos activates Ras by exchange of GTP for GDP. The RasGTP binds directly to a serine/threonine kinase, Raf, forming a transient membrane anchoring signal.6,8 Active Raf kinase phosphorylates a dual specificity kinase, MEK, on serine218 and serine222 and activates it.7 MEK can also be phosphorylated by Mos, a protein kinase expressed during meiotic maturation of oocytes and by MEKK1.8 A generally held belief is that MEK1 binds ERK and phosphorylates either a threonine or a tyrosine residue and then dissociates. The monophosphorylated ERK then rebinds to an active MEK1 for dual phosphorylation and complete activation.9,10 The activated MEK phosphorylates ERK1/ERK2 on threonine183 and tyrosine185 (at the TEY motif). The major targets of activated ERKs are pp90 ribosomal S6 kinase (Rsk) and the cytoplasmic phospholipase A2.11 ERK also translocates to the nucleus to phosphorylate the transcription factor Elk-1 (on serine383 and serine389). Recently another related kinase, ERK3, a nuclear protein kinase, has been cloned. It exhibits about 50% homology with ERK1/ERK2 within its catalytic domain.12 However, it does not phosphorylate any typical ERK substrates.
Usually only one highly active form of ERK1 or ERK2 (dual phosphorylated) exists in the cell, exhibiting over 1000-fold greater activity than the unphosphorylated form. Within the cell, at any time, one may find three less active forms of ERKs: one unphosphorylated enzyme, and two singly phosphorylated forms that contain phosphate either at the tyrosine or threonine residue.10 The JNK/SAPK (c-Jun kinase/stress activated protein kinase) cascade is activated following exposure to UV radiation, heat shock, or inflammatory cytokines.2,13 The activation of these MAP kinases is mediated by Rac and Cdc42, two small G-proteins.1,13 The activated Cdc42 binds to PAK65 protein kinase and activates it.14 The activated PAK65 can activate MEKK which in turn phosphorylates and activates SEK/ JNKK at serine219 and serine223.2 The active SEK/JNKK phosphorylates JNK/SAPK (at the TPY motif) that in turn binds to the Nterminal region of c-Jun and phosphorylates it at serine63 and serine73.16 The sites of activating phosphorylation are conserved between ERK and JNK, however, these sites are located within distinct dual specificity phosphorylation motifs (TPY for JNK and TEY for ERK).1,17 Molecular cloning studies show about 40 - 45% sequence homology between JNK/SAPK and the classical MAP kinases.16,18 The p38 kinase (reactivating kinase) is the newest member of the MAP kinase family.2 It is activated in response to inflammatory cytokines, endotoxins, and osmotic stress.5,18,19 It shares about 50% homology with the ERKs.5 The upstream steps in its activation are not well defined. However, downstream activation of p38 occurs following its phosphorylation (at the TGY motif) by MKK3, a dual specificity kinase. Following its activation, p38 translocates to the nucleus and phosphoryates ATF-2.20 Another known target of p38 is MAPKAPK2 that is involved in the phosphorylation and activation of heat-shock proteins.21