Molecular networks in FGF signaling: flotillin-1 and cbl-associated protein compete for the binding to fibroblast growth factor receptor substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.     

Vinexin, CAP/ponsin, ArgBP2: a novel adaptor protein family regulating cytoskeletal organization and signal transduction

Adaptor proteins, composed of two or more protein-protein interacting modules without enzymatic activity, regulate various cellular functions. Vinexin, CAP/ponsin, and ArgBP2 constitute a novel adaptor protein family. They have a novel conserved region homologous to the active peptide sorbin, as well as three SH3 (src homology 3) domains. A number of proteins binding to this adaptor family have been identified. There is accumulating evidence that this protein family regulates cell adhesion, cytoskeletal organization, and growth factor signaling. This review will summarize the structure and the function of proteins in this family.     

Regulation of transforming growth factor-beta signaling and PDK1 kinase activity by physical interaction between PDK1 and serine-threonine kinase receptor-associated protein

To gain more insights about the biological roles of PDK1, we have used the yeast two-hybrid system and in vivo binding assay to identify interacting molecules that associate with PDK1. As a result, serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor-beta (TGF-beta) receptor-interacting protein, was identified as an interacting partner of PDK1. STRAP was found to form in vivo complexes with PDK1 in intact cells. Mapping analysis revealed that this binding was only mediated by the catalytic domain of PDK1 and not by the pleckstrin homology domain. Insulin enhanced a physical association between PDK1 and STRAP in intact cells, but this insulin-induced association was prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. In addition, the association between PDK1 and STRAP was decreased by TGF-beta treatment. Analysis of the activities of the interacting proteins showed that PDK1 kinase activity was significantly increased by coexpression of STRAP, probably through the inhibition of the binding of 14-3-3, a negative regulator, to PDK1. Consistently, knockdown of the endogenous STRAP by the transfection of the small interfering RNA resulted in the decrease of PDK1 kinase activity. PDK1 also exhibited an inhibition of TGF-beta signaling with STRAP by contributing to the stable association between TGF-beta receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-beta. Knockdown of endogenous PDK1 with small interfering RNA has an opposite effect. Taken together, these results suggested that STRAP acts as an intermediate signaling molecule linking between the phosphatidylinositol 3-kinase/PDK1 and the TGF-beta signaling pathways.     

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.     

A functional interaction between the p75 neurotrophin receptor interacting factors, TRAF6 and NRIF

Neurotrophin signaling through the p75 receptor regulates apoptosis within the nervous system both during development and in response to injury. Whereas a number of p75 interacting factors have been identified, how these upstream factors function in a coordinated manner to mediate receptor signaling is still unclear. Here, we report a functional interaction between TRAF6 and the neurotrophin receptor interacting factor (NRIF), two proteins known to associate with the intracellular domain of the p75 neurotrophin receptor. The association between NRIF and TRAF6 was direct and occurred with both endogenous and ectopically expressed proteins. A KRAB repressor domain of NRIF and the carboxyl-terminal, receptor-binding region of TRAF6 were required for the interaction. Co-expression of TRAF6 increased the levels of NRIF protein and induced its nuclear translocation. Reciprocally, NRIF enhanced TRAF6-mediated activation of the c-Jun NH2-terminal kinase (JNK) by 3-fold, while only modestly increasing the stimulation of NF-kappaB. The expression of both NRIF and TRAF6 was required for reconstituting p75 activation of JNK in HEK293 cells, whereas NRIF mutants lacking the TRAF6 interaction domain were unable to substitute for the full-length protein in facilitating activation of the kinase. These results suggest that NRIF and TRAF6 functionally interact to facilitate neurotrophin signaling through the p75 receptor.     

The expression of teneurin-4 in the avian embryo: potential roles in patterning of the limb and nervous system

Teneurins are type II transmembrane proteins that play important roles in pattern formation in Drosophila, axon fasciculation and organogenesis in Caenorhabidits elegans, and neuronal pathfinding in the visual system of the mouse. There is evidence that a peptide derived from a proteolytic event near the C-terminus of teneurins leads to formation of an active neuropeptide, while processing at and near the transmembrane domain leads to shedding of the extracellular domain into the extracellular matrix and the generation of an intracellular fragment that is transported to the nucleus. In vertebrates there are four teneurins. Here, we have studied the expression of teneurin-4 in the chicken embryo. An antiserum against part of the intracellular domain of teneurin-4 recognizes several low molecular weight bands on immunoblots of embryonic chicken brain homogenates, indicating that teneurin-4 is likely to be processed at one or more predicted proteolytic cleavage sites. Antisera against the EGF-like repeats of the extracellular domain label some mesenchyme in the early embryo, and near basement membranes this labeling partially overlaps with anti-laminin (gamma 1 chain) immunostaining. At embryonic day 7, anti-teneurin-4 labels bundles of axons in the nasal, but not temporal retina. Later in development, retinal expression switches so that teneurin-4 is found in the temporal, but not nasal, ganglion cell layer. Teneurin-4 immunolocalization was also compared with other teneurins in the developing limb, where each teneurin is expressed in distinctive regions. These patterns of expression suggest roles for teneurin-4 in patterning and neuronal pathfinding in the avian embryo.     

Real-time imaging nuclear translocation of Akt1 in HCC cells

Akt is one of the critical mediators in cellular signaling, and overactivation of Akt related pathway frequently occurs in hepatocellular carcinoma (HCC). In this study, we presented that Akt was upregulated in HCC cell lines, and its active phosphorylated form was mainly located in the nucleus. Employing the laser confocal techniques for imaging intracellular protein dynamics, we monitored the transnuclear movement of GFP-tagged wild-type Akt1 (Akt1-WT-GFP) and its inactive mutant (Akt1-T308A/S473A-GFP) in live SMMC-7721 HCC cells, and both of fusion proteins were found to distribute over the cytoplasm and nucleus. Moreover, it was found that platelet derived growth factor (PDGF) was able to accelerate the nuclear translocation of wild-type Akt1 in HCC cells but failed to speed up the motion of the mutant. It was demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt1 facilitated the nuclear translocation of Akt1, but the phosphorylation at threonine 308 and serine 473 was not prerequisite.     

A functional nuclear localization sequence in the C-terminal domain of SHP-1

The Src homology 2 domain-containing protein tyrosine phosphatases SHP-1 and SHP-2 play an important role in many intracellular signaling pathways. Both SHP-1 and SHP-2 have been shown to interact with a diverse range of cytosolic and membrane-bound signaling proteins. Generally, SHP-1 and SHP-2 perform opposing roles in signaling processes; SHP-1 acts as a negative regulator of transduction in hemopoietic cells, whereas SHP-2 acts as a positive regulator. Intriguingly, SHP-1 has been proposed to play a positive regulating role in nonhemopoietic cells, although the mechanisms for this are not understood. Here we show that green fluorescent protein-tagged SHP-1 is unexpectedly localized within the nucleus of transfected HEK293 cells. In contrast, the highly related SHP-2 protein is more abundant within the cytoplasm of transfected cells. In accordance with this, endogenous SHP-1 is localized within the nucleus of several other nonhemopoietic cell types, whereas SHP-2 is distributed throughout the cytoplasm. In contrast, SHP-1 is confined to the cytoplasm of hemopoietic cells, with very little nuclear SHP-1 evident. Using chimeric SHP proteins and mutagenesis studies, the nuclear localization signal of SHP-1 was identified within the C-terminal domain of SHP-1 and found to consist of a short cluster of basic amino acids (KRK). Although the KRK motif resembles half of a bipartite nuclear localization signal, it appears to function independently and is absolutely required for nuclear import. Our findings show that SHP-1 and SHP-2 are distinctly localized within nonhemopoietic cells, with the localization of SHP-1 differing dramatically between nonhemopoietic and hemopoietic cell lineages. This implies that SHP-1 nuclear import is a tightly regulated process and indicates that SHP-1 may possess novel nuclear targets.     

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.     

Angiogenin activates Erk1/2 in human umbilical vein endothelial cells

Angiogenin is a potent angiogenic factor that binds to endothelial cells and is endocytosed and rapidly translocated to the nucleus where it is concentrated in the nucleolus and binds to DNA. Angiogenin also activates cell-associated proteases, induces cell invasion and migration, stimulates cell proliferation, and organizes cultured cells to form tubular structures. The intracellular signaling pathways that mediate these various cellular responses are not well understood. Here we report that angiogenin induces transient phosphorylation of extracellular signal-related kinase1/2 (Erk1/2) in cultured human umbilical vein endothelial cells. Angiogenin does not affect the phosphorylation status of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinases. PD98059--a specific inhibitor of MAP or Erk kinase 1 (MEK 1), the upstream kinase that phosphorylates Erk1/2--abolishes angiogenin-induced Erk phosphorylation and cell proliferation without affecting nuclear translocation of angiogenin. In contrast, neomycin, a known inhibitor of nuclear translocation and cell proliferation, does not interfere with angiogenin-induced Erk1/2 phosphorylation. These data indicate that both intracellular signaling pathways and direct nuclear functions of angiogenin are required for angiogenin-induced cell proliferation and angiogenesis.