Megan Robinson

Mitogen-activated protein kinase pathways.

Nearly all cell surface receptors utilize one or more of the mitogen-activated protein kinase cascades in their repertoire of signal transduction mechanisms. Recent advances in the study of such cascades include the cloning of genes encoding novel members of the cascades, further definition of the roles of the cascades in responses to extracellular signals, and examination of cross-talk between different cascades.

Phosphorylation of the MAP Kinase ERK2 Promotes Its Homodimerization and Nuclear Translocation

The MAP kinase ERK2 is widely involved in eukaryotic signal transduction. Upon activation it translocates to the nucleus of the stimulated cell, where it phosphorylates nuclear targets. We find that nuclear accumulation of microinjected ERK2 depends on its phosphorylation state rather than on its activity or on upstream components of its signaling pathway. Phosphorylated ERK2 forms dimers with phosphorylated and unphosphorylated ERK2 partners. Disruption of dimerization by mutagenesis of ERK2 reduces its ability to accumulate in the nucleus, suggesting that dimerization is essential for its normal ligand-dependent relocalization. The crystal structure of phosphorylated ERK2 reveals the basis for dimerization. Other MAP kinase family members also form dimers. The generality of this behavior suggests that dimerization is part of the mechanism of action of the MAP kinase family.

A constitutively active and nuclear form of the MAP kinase ERK2 is sufficient for neurite outgrowth and cell transformation.

BACKGROUND Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. RESULTS To create an activated ERK2 variant, we fused ERK2 to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein was active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells did not activate endogenous ERK1 or ERK2. It was sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1, neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein was localized to the nucleus. A cytoplasmic fusion protein was without effect. CONCLUSIONS Activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events.

Stress Pathway Activation Induces Phosphorylation of Retinoid X Receptor

Cellular stresses inhibit retinoid signaling, but the molecular basis for this phenomenon has not been revealed. Here, we present evidence that retinoid X receptor (RXR) is a substrate for both mitogen-activated protein kinase kinase-4 (MKK4/SEK1) and its downstream mediator c-Jun N-terminal kinase (JNK). MKK4/SEK1 and JNK recognized distinct features on RXR in the DE and AB regions, respectively. Phosphorylation by MKK4/SEK1 had profound effects on the biochemical properties of RXR, inhibiting the expression of genes activated by RXR-retinoic acid receptor complexes. Tyr-249 in the RXR DE region was required for the inhibitory effect of MKK4/SEK1. These effects were significantly reduced in MKK4/SEK1-null cells, indicating that MKK4/SEK1 is required for the suppression of retinoid signaling by stress. Findings presented here demonstrate that MKK4/SEK1 can directly modulate transcription by phosphorylating RXR, a novel MKK4/SEK1 substrate.

Contributions of the Mitogen-activated Protein (MAP) Kinase Backbone and Phosphorylation Loop to MEK Specificity

To examine the specificity of MEKs for MAP kinase family members, we determined the abilities of several MEK isoforms to phosphorylate mutants of the MAP kinase ERK2 and the related kinase ERK3 which are modified in the phosphorylation loop. The ERK2 mutants included mutations of the two phosphorylation sites, mutations of the acidic residue between these two sites, and mutations that shorten the length of this loop. All mutants were tested for phosphorylation by six mammalian MEKs and compared with several wild type MAP kinases. MEK1 and MEK2 phosphorylate a majority of the ERK2 mutants. MEK2 but not MEK1 will phosphorylate ERK3. Alteration of the residue between the two phosphorylation sites neither dramatically affected the activity of MEK1 and MEK2 toward ERK2 nor conferred recognition by other MEKs. Likewise, reduction of the length of the phosphorylation loop only partially reduces recognition by MEK1 and MEK2 but does not promote recognition by other MEKs. Thus other yet to be identified factors must contribute to the specificity of MEK recognition of MAP kinases.

Myogenic Differentiation of Muscular Dystrophy-Specific Induced Pluripotent Stem Cells for Use in Drug Discovery

Human induced pluripotent stem cells (iPSCs) represent a scalable source of potentially any cell type for disease modeling and therapeutic screening. We have a particular interest in modeling skeletal muscle from various genetic backgrounds; however, efficient and reproducible methods for the myogenic differentiation of iPSCs have not previously been demonstrated. Ectopic myogenic differentiation 1 (MyoD) expression has been shown to induce myogenesis in primary cell types, but the same effect has been unexpectedly challenging to reproduce in human iPSCs. In this study, we report that optimization of culture conditions enabled direct MyoD‐mediated differentiation of iPSCs into myoblasts without the need for an intermediate step or cell sorting. MyoD induction mediated efficient cell fusion of mature myocytes yielding multinucleated myosin heavy chain‐positive myotubes. We applied the same approach to dystrophic iPSCs, generating 16 iPSC lines from fibroblasts of four patients with Duchenne and Becker muscular dystrophies. As seen with iPSCs from healthy donors, within 36 hours from MyoD induction there was a clear commitment toward the myogenic identity by the majority of iPSCs in culture (50%–70%). The patient iPSC‐derived myotubes successfully adopted the skeletal muscle program, as determined by global gene expression profiling, and were functionally responsive to treatment with hypertrophic proteins insulin‐like growth factor 1 (IGF‐1) and wingless‐type MMTV integration site family, member 7A (Wnt7a), which are being investigated as potential treatments for muscular dystrophy in clinical and preclinical studies, respectively. Our results demonstrate that iPSCs have no intrinsic barriers preventing MyoD from inducing efficient and rapid myogenesis and thus providing a scalable source of normal and dystrophic myoblasts for use in disease modeling and drug discovery.

A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs

Human induced pluripotent stem cells (hiPSCs) hold enormous potential, however several obstacles impede their translation to industrial and clinical applications. Here we describe a platform to efficiently generate, characterize and maintain single cell and feeder-free (FF) cultured hiPSCs by means of a small molecule cocktail media additive. Using this strategy we have developed an effective multiplex sorting and high-throughput selection platform where individual clonal hiPSC lines are readily obtained from a pool of candidate clones, expanded and thoroughly characterized. By promoting survival and self-renewal, the selected hiPSC clones can be rapidly expanded over multiple FF, single-cell passages while maintaining their pluripotency and genomic stability as demonstrated by trilineage differentiation, karyotype and copy number variation analysis. This study provides a robust platform that increases efficiency, throughput, scale and quality of hiPSC generation and facilitates the industrial and clinical use of iPSC technology.

Characterization of a Protein Kinase that Phosphorylates Serine 189 of the Mitogen-activated Protein Kinase Homolog ERK3

A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser, comparable to Thr, one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members.

Structural Analysis of the MAP Kinase ERK2 and Studies of MAP Kinase Regulatory Pathways

Publisher Summary This chapter primarily highlights structure–function studies performed collaboratively between the Goldsmith and Cobb laboratories at the University of Texas Southwestern Medical Center in Dallas and includes experiments examining the relationship of MEKKl to the MAP kinase pathway and potential feedback mechanisms in the pathway. Mechanisms regulating the MAP kinase pathway are complicated and inactidvation methods plentiful. Receptor tyrosine kinases regulate the pathway through Ras; heterotrimeric G protein-coupled receptors also use Ras to activate the pathway, although there may also be Ras-independent mechanisms. The MAP kinase pathway is stimulated by numerous hormones, growth factors, and oncogene products including Ras and contributes to their spectrum of actions. The MAP kinases, however, are pleiotropic, phosphorylating many substrates throughout the cell. This pathway has been repeated several times in yeast and mammalian cells, although mechanisms regulating the similar but parallel cascades are sketchier. There are three low-activity forms of each enzyme, the unphosphorylated protein and the two singly phosphorylated forms, that contain phosphate on only tyrosine or only threonine. These two singly phosphorylated ERKs have little more protein kinase activity than unphosphorylated proteins. Because ERKl and ERK2 can autophosphorylate on tyrosine, the form containing only threonine phosphate may, in as yet undefined circumstances, be able to reactivate itself through autophosphorylation. The three-dimensional structure of ERK2 contains the two-domain organization found in all protein kinases whose structures have been determined thus far. The smaller N-terminal domain provides many contacts for ATP, and the larger C-terminal domain contains the major determinants for protein substrate interactions. It is very important to identify the unique features, if any, of these enzyme pairs. It is interesting that phosphorylation by MAP kinase had so little effect on MEK activity, because phosphorylation of MEKl by cdc2 at a site (S286) close to the MAP kinase site (T292) in the C-terminal insert greatly inhibits MEKl activity.

Optimized Surface Markers for the Prospective Isolation of High-Quality hiPSCs using Flow Cytometry Selection

hiPSC derivation and selection remains inefficient; with selection of high quality clones dependent on extensive characterization which is not amenable to high-throughput (HTP) approaches. We recently described the use of a cocktail of small molecules to enhance hiPSC survival and stability in single cell culture and the use of flow cytometry cell sorting in the HTP-derivation of hiPSCs. Here we report an enhanced protocol for the isolation of bona fide hiPSCs in FACS-based selection using an optimized combination of cell surface markers including CD30. Depletion of CD30+ cells from reprogramming cultures almost completely abolished the NANOG and OCT4 positive sub-population, suggesting it is a pivotal marker of pluripotent cells. Combining CD30 to SSEA4 and TRA-1-81 in FACS greatly enhanced specificity and efficiency of hiPSC selection and derivation. The current method allows for the efficient and automated, prospective isolation of high-quality hiPSC from the reprogramming cell milieu.