Structural Studies Of Signal Transduction Mechanisms In The Mitogen-activated Protein Kinase Pathways

Mitogen - activated protein kinase (MAPK) signaling modules are one of the most widespread signaling systems in eukaryotes that transfer signals from the cell surface to the nucleus, controlling multiple cellular programs such as em-bryogenesis, cell differentiation, cell proliferation, and cell death. Each MAPK pathway consists of a central three - tiered protein kinases in which MAPKs are activated by dual phosphorylation on a motif Thr - X - Tyr catalyzed by a family of dual specificity kinases known as MAPK kinases (MAP2Ks) or MAP/extracellular signal - regulated kinase (ERK) kinases (MEKs). MAP2Ks, in turn, are activated by a protein kinase superfamily referred to as MAPK kinase kinases (MAP3Ks) or MEK kinases (MEKKs). Finally, activated MAPKs phosphoryl-ate various substrates in cytoplasm and nucleus to change the cellular program and to regulate gene expression pattern. Inactivation of MAP kinases is achieved by dephosphorylation of either or both pThr and pTyr by different classes of protein phosphatases, tyrosine phosphatases (PTPs) , serine/threonine - phospha-tases, or dual specificity phosphatases (MAP kinase phosphatases, or MKPs).Among the twelve or more homologous mammalian MAPKs that have been i-dentified, three, the ERKs, p38s, and JNKs (c - Jun N -terminal kinases) have become well - studied. ERKs are activated by mitogenic stimuli, such as hormones, growth factors, and phorbol esters, and thus are associated with pro-liferative processes. By contrast, p38s and JNKs are more potently activated in response to physical and chemical stresses such as osmotic shock, UV radiation, oxidative stress, and inflammatory cytokines, and are linked both to reparativeand apoptotic responses. Given that there are many MAP kinases functioning in different MAP kinase pathways by interacting with different proteins, how specificity is conferred remains an important unsolved problem.TAO2 is a MAP3K level kinase, catalyzing the activating double phospho-rylation on the MAP2Ks MEK3 and MEK6 towards p38s. In order to gain insights for the three - dimensional structure of MAP3 K level kinases, we have determined the crystal structure of TA02 kinase domain, a Ste20p related MAP3K in the stress - responsive p38 MAPK pathway. In addition, the interactions between MAPKs and their substrates, activating kinases and down - regulating phosphatases are known to be regulated by docking interaction, which is different from the interactions at the active sites of MAPKs. We have also determined the crystal structure of low activity ERK2 in complex with a docking motif pep-tide ( pepHePTP) derived from hematopoietic protein tyrosine phosphatase ( HePTP) , a negative regulator of ERK2.MATERIALS AND METHODSMATERIALS1. For the crystal structure of TA02 kinase domain (1 - 320) : Bac - To -Bac Baculovims Expression Systems (life Technologies, USA) for cloning of TAO2 (1 - 320) into baculovirus;Hi5 insect cells for protein expression;Ak-taFPLC systems ( pharmacia amersham, USA) for protein purification;and crystallization condition;0. 1 M imidazole pH 8. 0, 20% PEG 1000, and 0.2 M calcium acetate for the native protein, and 0.1 M sodium cacodylate, pH 6.5, 10% PEG 3000, and 0. 2 M magnesium chloride for the selenomethionine derivative protein.2. For the crystal structure of inactive ERK2 in complex with pepHePTP: Vector NpT7 - 5 as expression vector for the full length rat ERK2;bacteria Rosetta cells for the protein expression;AktaFPLC systems (pharmacia amersham , USA) for protein purification;D - domain docking peptide pepHePTP derived from phosphatase HePTP with sequence 16RLQERRGSNVALMLDV31 synthesized, and mixed with ERK2 to make the complex, and crystallizationcondition: 0.1 M HEPES pH 7.5, 20% PEG 4000, and 10% isopropanol. METHODS1. For the crystal structure of TA02 kinase domain (1 - 320) : infection of Hi5 insect cells with recombinant baculovirus carrying TAO2 (1 - 320) gene to express the TAO2 kinase domain protein;affinity, ion exchange, and size exclusion columns for protein purification;hanging drop vapor diffusion method for growing crystals;and SAD phasing method for structural determination.2. For the crystal structure of low activity ERK2 in complex with pepHeP-TP: transformation of NpT7 - 5 plasmid carrying rat ERK2 gene into bacteria Rosseta cells for protein expression;affinity and ion exchange columns for protein purification;hanging drop vapor diffusion method for growing complex crystals between ERK2 and pepHePTP;and molecular replacement method for structural determination.RESULTS1. The crystal structure of TA02 kinase domain (1 - 320) was determined at 2. 0A resolution. The TAO2 kinase domain purified from insect cells was con-stitutively active, and the structure reveals phosphorylation on serinel81 at the activation loop of the kinase, and thereby adopts a closed active conformation. TAO2 (1 — 320) possesses the typical protein kinase two domain architecture with two additional helices in both the N - and C - terminal extensions. The N -terminal domain is composed of the standard five - stranded (3 sheet and the a helix C, and the C - terminal domain possesses six major helices, two (3 - ribbons, the catalytic loop, and the activation loop. Phosphoserinel81 at the activation loop acts as an organizing center to align the catalytically important residues into correct positions, thus making an active kinase. The phosphorylation - dependent activation of TAO2 involves massive refolding of the activation loop including the P +1 specificity pocket. The structure also reveals a hydrophobic P + 1 pocket, comprised of Phel82, Prol86, Metl89 (from the C - terminal portion of the activation loop) and Ler234 (from helix G) , which was shown by mutagenic studies to involve in the substrate MEK6 recognition. A positively -charged pocket, consisting of Lysl20, Arg221 and Lys222, has been identified by model building, and was shown to contribute to the phosphorylation of MEK6. The structure of MgATP - bound TA02 (1-320) reveals unique feature in the nucleotide binding, involving, in part, the C - terminal extension of the kinase;Lys314 from helix K forms a hydrogen bond with the 2'hydroxyl of ribose of ATP.2. The crystal structure of low activity ERK2 in complex with a docking motif peptide (pepHePTP) has been determined at 1.9 A resolution. pepHeP-TP, with sequence 16RLQERRGSNVALMLDV31, conforms to the consensus docking motif sequence ( Arg/Lys) 2 - (X) 2 ₆ - $> A - X - (p B, where $ A and