Identification of the T-complex protein as a binding partner for newly synthesized cytoplasmic dynein intermediate chain 2.

Cytoplasmic dynein is a macromolecular motor complex with diverse functions in eukaryotic cells. Dynein plays essential roles in intracellular transport of organelles and mitosis, mediated in part by interactions between the dynein intermediate chain 2 (IC-2) subunits and adapter proteins that bind specific cargos. In experiments to identify phosphorylation-dependent binding partners for IC-2 we instead identified a phosphorylation-independent binding partner, the cytosolic chaperonin containing T complex protein 1 (CCT).

Epidermal growth factor stimulates extracellular-signal regulated kinase phosphorylation of a novel site on cytoplasmic Dynein intermediate chain 2.

Extracellular-signal regulated kinase (ERK) signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2) as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts.

The Salmonella kinase SteC targets the MAP kinase MEK to regulate the host actin cytoskeleton.

After host cell entry, Salmonella replicate in membrane-bound compartments, which accumulate a dense meshwork of F-actin through the kinase activity of the Salmonella SPI-2 type III secretion effector SteC. We find that SteC promotes actin cytoskeleton reorganization by activating a signaling pathway involving the MAP kinases MEK and ERK, myosin light chain kinase (MLCK) and Myosin IIB. Specifically, SteC phosphorylates MEK directly on serine 200 (S200), a previously unstudied phosphorylation site.

Trk activation of the ERK1/2 kinase pathway stimulates intermediate chain phosphorylation and recruits cytoplasmic dynein to signaling endosomes for retrograde axonal transport.

The retrograde transport of Trk-containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phosphoserine on the dynein intermediate chains (ICs), and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types.

Extracellular signal-regulated kinase promotes Rho-dependent focal adhesion formation by suppressing p190A RhoGAP.

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood.

Gonadal hormones modulate the potency of the disruptive effects of donepezil in male rats responding under a nonspatial operant learning and performance task.

In contrast to estrogen in female rats, testosterone in male rats may decrease cholinergic activity in the brain, thereby attenuating behaviors mediated by the cholinergic system. To investigate this possibility, the interactive effects of the gonadal hormones and donepezil, an acetylcholinesterase (AChE) inhibitor, on the responding of male rats were examined under a multiple schedule of repeated acquisition and performance of response sequences and on AChE activity in specific brain regions. Donepezil dose-effect curves (0.

Differential requirement for MEK Partner 1 in DU145 prostate cancer cell migration.

ERK signaling regulates focal adhesion disassembly during cell movement, and increased ERK signaling frequently contributes to enhanced motility of human tumor cells. We previously found that the ERK scaffold MEK Partner 1 (MP1) is required for focal adhesion disassembly in fibroblasts. Here we test the hypothesis that MP1-dependent ERK signaling regulates motility of DU145 prostate cancer cells.

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: a perspective.

Cell migration is critical for many physiological processes and is often misregulated in developmental disorders and pathological conditions including cancer and neurodegeneration. MAPK signaling and the Rho family of proteins are known regulators of cell migration that exert their influence on cellular cytoskeleton during cell adhesion and migration. Here we review data supporting the view that localized ERK signaling mediated through recently identified scaffold proteins may regulate cell migration.

MEK1 activation by PAK: a novel mechanism.

Extracellular signal-Regulated Kinase (ERK) controls a variety of cellular processes, including cell proliferation and cell motility. While oncogenic mutations in Ras and B-Raf result in deregulated ERK activity and proliferation and migration in some tumor cells, other tumors exhibit elevated ERK signaling in the absence of these mutations. Here we provide evidence that PAK can directly activate MEK1 by a mechanism distinct from conventional Ras/Raf mediated activation.

The MEK1 scaffolding protein MP1 regulates cell spreading by integrating PAK1 and Rho signals.

How the extracellular signal-regulated kinase (ERK) cascade regulates diverse cellular functions, including cell proliferation, survival, and motility, in a context-dependent manner remains poorly understood. Compelling evidence indicates that scaffolding molecules function in yeast to channel specific signals through common components to appropriate targets. Although a number of putative ERK scaffolding proteins have been identified in mammalian systems, none has been linked to a specific biological response.

MEK partner 1 (MP1): regulation of oligomerization in MAP kinase signaling.

Specificity in signal transduction can be achieved through scaffolds, anchors, and adapters that assemble generic signal transduction components in specific combinations and locations. MEK Partner-1 (MP1) was identified as a potential "scaffold" protein for the mammalian extracellular signal-regulated kinase (ERK) pathway. To gain insight into the interactions of MP1 with the ERK pathway, we analyzed the ability of MP1 to bind to MEK1, ERK1, and to itself, and the regulation of these interactions.

Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion.

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1.

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation.

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation.

Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes.

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac.

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites.

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry.

Novel activation of STAT5b in response to epidermal growth factor.

Of the seven signal transducers and activators of transcription that have been identified, STATs 1, 3, and 5a/5b can be activated not only by a multitude of cytokines but also by some growth factors. The data presented here demonstrate that, in contrast to activation by the cytokine, growth hormone (GH), the activation of STAT5b by the growth factor, epidermal growth factor (EGF), requires overexpression of the EGF receptor (EGFR). We have shown that EGF activates STAT5b not only in a HEK293 cell model in which the EGFR is stably overexpressed but also in the MDA-MB468 breast cancer cell line.