07-531 Sigma-AldrichAnti-PIR121-1/Sra-1 Antibody

Although the MAP kinase-interacting kinases (MNKs) have been known for greater than 15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

Axonal branching and synapse formation are tightly linked developmental events during the establishment of synaptic circuits. Newly formed synapses promote branch initiation and stability. However, little is known about molecular mechanisms that link these two processes. Here, we show that local assembly of an F-actin cytoskeleton at nascent presynaptic sites initiates both synapse formation and axon branching. We further find that assembly of the F-actin network requires a direct interaction between the synaptic cell adhesion molecule SYG-1 and a key regulator of actin cytoskeleton, the WVE-1/WAVE regulatory complex (WRC). SYG-1 cytoplasmic tail binds to the WRC using a consensus WRC interacting receptor sequence (WIRS). WRC mutants or mutating the SYG-1 WIRS motif leads to loss of local F-actin, synaptic material, and axonal branches. Together, these data suggest that synaptic adhesion molecules, which serve as a necessary component for both synaptogenesis and axonal branch formation, directly regulate subcellular actin cytoskeletal organization.

Neuronal morphology is highly sensitive to ischemia, although some re-organization may promote neuroprotection. In this study we investigate the role of actin regulating proteins (ARP2, ARP3 and WAVE-1) and their role in rapid ischemic tolerance. Using an established in vitro model of rapid ischemic tolerance, we show that WAVE-1 protein levels are stabilized following brief tolerance inducing ischemia (preconditioning). The stabilization appears to be due to a reduction in the ubiquitination of WAVE-1. Levels of ARP2, ARP3 and N-WASP were not affected by ischemic preconditioning. Immunocytochemical studies show a relocalization of ARP2 and ARP3 proteins in neurons following preconditioning ischemia, as well as a re-organization of actin. Blocking the protein kinase CK2 using emodin blocks ischemic tolerance, and our data suggests CK2 binds to WAVE-1 in neurons. We observe an increase in binding of the ARP2 subunit with WAVE-1. The neuroprotection observed following preconditioning is inhibited when cells are transduced with an N-WASP CA domain that blocks the activation of ARP2/3. Together these data show that ischemia affects actin regulating enzymes, and that the ARP2/3 pathway plays a role in rapid ischemic tolerance induced neuroprotection.

The seven-subunit ARP2/3 complex is an efficient modulator of the actin cytoskeleton with well-recognized roles in amoeboid locomotion and subcellular motility of organelles and microbes. The recent identification of different subunit homologs suggests the existence of a functional ARP2/3 complex in higher plants. Mutations in some of the subunits have revealed a pivotal role for the complex in determining the shape of walled cells and focused attention on the interlinked processes of cortical-actin organization, growth-site selection, organelle motility and actin-microtubule interactions during plant cell morphogenesis. The findings supporting a global conservation of molecular mechanisms for membrane protrusion have been further strengthened by the identification of plant homologs of upstream regulators of the complex such as PIR121, NAP125 and HSPC300. As discussed here, the recent studies suggest that there might be hitherto unappreciated molecular and cell-biological commonalities between protrusion mediated motility of animal cells and polarized, expansion-mediated growth of plant cells.

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.

Tuba is a novel scaffold protein that functions to bring together dynamin with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Tuba binds a variety of actin regulatory proteins, including N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1, and Ena/VASP proteins, via a C-terminal SH3 domain. Direct binding partners include N-WASP and Ena/VASP proteins. Forced targeting of the C-terminal SH3 domain to the mitochondrial surface can promote accumulation of F-actin around mitochondria. A Dbl homology domain present in the middle of Tuba upstream of a Bin/amphiphysin/Rvs (BAR) domain activates Cdc42, but not Rac and Rho, and may thus cooperate with the C terminus of the protein in regulating actin assembly. The BAR domain, a lipid-binding module, may functionally replace the pleckstrin homology domain that typically follows a Dbl homology domain. The properties of Tuba provide new evidence for a close functional link between dynamin, Rho GTPase signaling, and the actin cytoskeleton.

BACKGROUND: In animal cells, GTPase signaling pathways are thought to generate cellular protrusions by modulating the activity of downstream actin-regulatory proteins. Although the molecular events linking activation of a GTPase to the formation of an actin-based process with a characteristic morphology are incompletely understood, Rac-GTP is thought to promote the activation of SCAR/WAVE, whereas Cdc42 is thought to initiate the formation of filopodia through WASP. SCAR and WASP then activate the Arp2/3 complex to nucleate the formation of new actin filaments, which through polymerization exert a protrusive force on the membrane. RESULTS: Using RNAi to screen for genes regulating cell form in an adherent Drosophila cell line, we identified a set of genes, including Abi/E3B1, that are absolutely required for the formation of dynamic protrusions. These genes delineate a pathway from Cdc42 and Rac to SCAR and the Arp2/3 complex. Efforts to place Abi in this signaling hierarchy revealed that Abi and two components of a recently identified SCAR complex, Sra1 (p140/PIR121/CYFIP) and Kette (Nap1/Hem), protect SCAR from proteasome-mediated degradation and are critical for SCAR localization and for the generation of Arp2/3-dependent protrusions. CONCLUSIONS: In Drosophila cells, SCAR is regulated by Abi, Kette, and Sra1, components of a conserved regulatory SCAR complex. By controlling the stability, localization, and function of SCAR, these proteins may help to ensure that Arp2/3 activation and the generation of actin-based protrusions remain strictly dependant on local GTPase signaling.

p53 mutants in tumours have a reduced affinity for DNA and a reduced ability to induce apoptosis. We describe a mutant with the opposite phenotype, an increased affinity for some p53-binding sites and an increased ability to induce apoptosis. The apoptotic function requires transcription activation by p53. The mutant has an altered sequence specificity and selectively fails to activate MDM2 transcription. Loss of MDM2 feedback results in overexpression of the mutant, but the mutant kills better than wild-type p53 even in MDM2-null cells. Thus the apoptotic phenotype is due to a combination of decreased MDM2 feedback control and increased or unbalanced expression of other apoptosis-inducing p53 target genes. To identify these genes, DNA chips were screened using RNA from cells expressing the apoptosis-inducing mutant, 121F, and a sequence-specificity mutant with the reciprocal phenotype, 277R. Two potential new mediators of p53-dependent apoptosis were identified, Rad and PIR121, which are induced better by 121F than wild-type p53 and not induced by 277R. The 121F mutant kills untransformed MDM2-null but not wild-type mouse embryo fibroblasts and kills tumour cells irrespective of p53 status. It may thus expand the range of tumours which can be treated by p53 gene therapy.