David Robbins

ERKs: A family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation

Interaction with SV40 small tumor antigen (small t) compromised the ability of multimeric protein phosphatase 2A to inactivate the mitogen-activated protein kinase ERK1 and the mitogen-activated protein kinase kinase MEK1. Transient expression of small t in CV-1 cells activated MEK and ERK but did not affect Raf activity. Small t stimulated the growth of quiescent CV-1 cells almost as effectively as did serum. Coexpression of kinase-deficient ERK2 blocked most, but not all, of the proliferation caused by small t. Activation of the mitogen-activated protein kinase pathway and stimulation of cell growth were dependent on the interaction of small t with protein phosphatase 2A. These findings indicate that SV40 small t is capable of inducing cell growth through blockade of protein phosphatase and deregulation of the mitogen-activated protein kinase cascade.

Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2.

The hedgehog gene of Drosophila melanogaster encodes a secreted protein (HH) that plays a vital role in cell fate and patterning. Here we describe a protein complex that mediates signal transduction from HH. The complex includes the products of at least three genes: fused (a protein-serine/threonine kinase), cubitus interruptus (a transcription factor), and costal2 (a kinesin-like protein). The complex binds with great affinity to microtubules in the absence of HH, but binding is reversed by HH. Mutations in the extracatalytic domain of FU abolish both the biological function of the protein and its association with COS2. We conclude that the complex may facilitate signaling from HH by governing access of the cubitus interruptus protein to the nucleus.

ERKs, extracellular signal-regulated MAP-2 kinases.

A family of protein kinases, known alternatively as microtubule-associated protein-2/myelin basic protein kinases or extracellular signal-regulated kinases, is activated by numerous hormones, growth factors and other extracellular stimuli. At least two members of this family function as intermediate kinases in protein phosphorylation cascades. Their mechanisms of activation may involve autophosphorylation, which occurs on both threonine and tyrosine residues.

Map kinases ERK1 and ERK2: Pleiotropic enzymes in a ubiquitous signaling network

Publisher Summary MAP kinase is used to refer to at least two distinct proteins, known as the extracellular signal-regulated protein kinases ERK 1 and ERK2. The discussion of proteins that are regulated by ERKs points to the pleiotropic nature of signaling pathways regulated by this family of protein kinases. Relatively, few data are yet in hand to prove that any of these proteins are substrates for these enzymes in vivo. The impact of these enzymes on cell function can be deduced from the recent experiments using mutated enzymes. The ERKl phosphorylation site and lysine mutants have proved useful dominant inhibitors. In Jurkat cells, the mutants inhibit induction of the cytokine IL-2. It was found that ERKl and ERK2 mutants block the ability of ras, serum, and phorbol ester to induce transcription from a TPA response element. ERK2 mutants prevent proliferation caused by activated Raf EGF or small tau antigen. The ubiquitous MAP kinases are activated by a remarkable variety of hormones in differentiated cells and growth factors in dividing cells. Their activation has been linked to the transition from G0 to G1 in the cell cycle and to the induction of differentiated phenotypes. These enzymes are essential components of a universal protein kinase cascade implicated in the control of many cellular processes.

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.

Identification of an activator of the microtubule-associated protein 2 kinases ERK1 and ERK2 in PC12 cells stimulated with nerve growth factor or bradykinin.

Treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal‐regulated kinases ERK1 and ERK2, two isozymes of microtubule‐associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyro‐sine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42‐ (ERK1) and 44‐kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide‐binding protein‐coupled receptors are both capable of regulating these pathways.

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.

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.

Extracellular Signal-Regulated Protein Kinases (ERKS) 1, 2, and 3

Extracellular signal-regulated kinases (ERKs)1–4 are protein kinases participating in numerous signal transduction pathways. The two best studied of these, ERK1 and ERK2, are known as MAP kinases and were first identified as 43 and 41 kDa, hormonally stimulated enzymes that used microtubule-associated protein-2 and myelin basic protein as substrates 5–8 in addition to these two proteins they recognize a varied group of substrates many of which are known to have important regulatory functions, including protein kinases 9–12, membrane receptors 3, 14, transcriptional factors 15, 16 and cytoskeletal proteins 1–19, indicating a pleiotropic mode of action. It has been suggested that these enzymes cause aberrant phosphorylation of tau that occurs in Alzheimer’s disease. Among the earliest studies of these enzymes were ones documenting their activation by insulin in terminally differentiated 3T3-L1 adipocytes as well as ones demonstrating that they are activated at the transition of cells from G0 to G1 5, 6, 20–26, 33