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PubMedDate not supplied

Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. preliminary functional investigations.

Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation. Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayers of unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides were synthesized on the solid phase using N,N',N'-tri(tert-butyloxycarbonyl)-hydrazino acetic acid and further coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields were measured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology proved superior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields, implementation, and reproducibility. To check whether the grafted-peptides were accessible and functional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), which is involved in intracellular membrane trafficking, have been selected. Using this model, we demonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with the cytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting in cells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared by hydrazone chemoselective ligation, as a tool for biological investigation.

Adaptor Protein Complex 3↗

PubMed1995

Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit.

The high affinity receptor for GM-CSF consists of a unique alpha subunit and a beta subunit that is shared with receptors for IL-3 and IL-5. Activation of GM-CSF receptor (GMR) triggers two distinct cytoplasmic signalling pathways, JAK2 and Ras, and is sufficient to maintain proliferation of growth factor-dependent cell lines. Shc proteins are phosphorylated upon activation of GMR and may be involved in the transmission of GM-CSF signals to Ras. To define the role of Shc proteins in cells stimulated with GM-CSF, we investigated both the network of interactions that involve Shc after GM-CSF stimulation and the effects of overexpressing Shc proteins on the proliferative response to GM-CSF. Two cytoplasmic complexes, Grb2/Sos and Grb2/p140 bind through the Grb2 SH2 domain to phosphorylated Shc, and are thereby recruited to the beta subunit. Both complexes are stable, even in the absence of ligand, and depend on the direct association of p140 and Sos respectively with the SH3 domains of Grb2. p140 is an uncharacterized protein constitutively phosphorylated on tyrosine and, in its Grb2-bound form, expressed only in hematopoietic cells, the oligomeric complex formed by phosphorylated beta subunit-phosphorylated Shc-Grb2-SoS-p140 is also induced by IL-3 and L-5 stimulation of growth-factor dependent cell lines. Overexpression of wild-type Shc proteins in growth factor-dependent cells increases both MAP kinase activation and proliferation in response to GM-CSF. These effects require the association of Shc with Grb2. Taken together these results indicate that phosphorylation of Shc proteins is a crucial step in the transmission of GM-CSF proliferative stimuli, since it creates a high affinity binding site for the Grb2/SoS complex, whose function is to activate Ras and, for the Grb2/p140 complex, whose function remains unknown.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Mechanisms of resistance to TRAIL-induced apoptosis in cancer.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anticancer agent. However, considerable numbers of cancer cells, especially some highly malignant tumors, are resistant to apoptosis induction by TRAIL, and some cancer cells that were originally sensitive to TRAIL-induced apoptosis can become resistant after repeated exposure (acquired resistance). Understanding the mechanisms underlying such resistance and developing strategies to overcome it are important for the successful use of TRAIL for cancer therapy. Resistance to TRAIL can occur at different points in the signaling pathways of TRAIL-induced apoptosis. Dysfunctions of the death receptors DR4 and DR5 due to mutations can lead to resistance. The adaptor protein Fas-associated death domain (FADD) and caspase-8 are essential for assembly of the death-inducing signaling complex, and defects in either of these molecules can lead to TRAIL resistance. Overexpression of cellular FADD-like interleukin-1beta-converting enzyme-inhibitory protein (cFLIP) correlates with TRAIL resistance in several types of cancer. Overexpression of Bcl-2 or Bcl-X(L), loss of Bax or Bak function, high expression of inhibitor of apoptosis proteins, and reduced release of second mitochondria-derived activator of caspases (Smac/Diablo) from the mitochondria to the cytosol have all been reported to result in TRAIL resistance in mitochondria-dependent type II cancer cells. Finally, activation of different subunits of mitogen-activated protein kinases or nuclear factor-kappa B can lead to development of either TRAIL resistance or apoptosis in certain types of cancer cells.

Apoptosis↗

PubMed1996

Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association.

Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).

Amino Acid Sequence↗

PubMed2002

Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family.

The Toll/interleukin-1 (IL-1) receptor (TIR) family comprises two groups of transmembrane proteins, which share functional and structural properties. The members of the IL-1 receptor (IL-1R) subfamily are characterized by three extracellular immunoglobulin (Ig)-like domains. They form heterodimeric signaling receptor complexes consisting of receptor and accessory proteins. The members of the Toll-like receptor (TLR) subfamily recognize alarm signals that can be derived either from pathogens or the host itself. TLRs possess leucine-rich repeats in their extracellular part. TLRs can form dimeric receptor complexes consisting of two different TLRs or homodimers in the case of TLR4. The TLR4 receptor complex requires supportive molecules for optimal response to its ligand lipopolysaccharide (LPS). A hallmark of the TIR family is the cytoplasmic TIR domain that is indispensable for signal transduction. The TIR domain serves as a scaffold for a series of protein-protein interactions which result in the activation of a unique signaling module consisting of MyD88, interleukin-1 receptor associated kinase (IRAK) family members and Tollip, which is used exclusively by TIR family members. Subsequently, several central signaling pathways are activated in parallel, the activation of NFkappaB being the most prominent event of the inflammatory response. Recent developments indicate that in addition to the common signaling module MyD88/IRAK/Tollip, other molecules can modulate signaling by TLRs, especially of TLR4, resulting in differential biological answers to distinct pathogenic structures. Subtle differences in TLR signaling pathways are now becoming apparent, which reveal how the innate immune system decides at a very early stage the direction in which the adaptive immune response will develop. The creation of pathogen-specific mediator environments by dendritic cells defines whether a cellular or humoral response will be activated in response to the pathogen.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The role of protein kinase A anchoring via the RII alpha regulatory subunit in the murine immune system.

Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RIIalpha regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RIIalpha regulatory subunit of PKA (-/-) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and -/- mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the -/- mice are similar to WT controls. In vitro, splenocytes from -/- mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8(+) IFN-gamma producing effector cells were stimulated upon infection of WT or -/- mice with Listeria monocytogenes. These data represent the first study of the role of RIIalpha in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RIIalpha -/- mice, suggesting either that RIIalpha is not required for normal immune function or that other proteins are able to compensate for RIIalpha function.

A Kinase Anchor Proteins↗

PubMed2003

[Screening of new binding partners of CIKS with yeast two-hybrid system].

The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2003

14-3-3 zeta mediates integrin-induced activation of Cdc42 and Rac. Platelet glycoprotein Ib-IX regulates integrin-induced signaling by sequestering 14-3-3 zeta.

Integrin-induced cytoskeletal reorganizations are initiated by Cdc42 and Rac1 but little is known about mechanisms by which integrins activate these Rho GTPases. 14-3-3 proteins are adaptors implicated in binding and regulating the function and subcellular location of numerous signaling molecules. In platelets, the 14-3-3 zeta isoform interacts with the glycoprotein (GP) Ibalpha subunit of the adhesion receptor GP Ib-IX. In this study, we show that integrin-induced activation of Cdc42, activation of Rac, cytoskeletal reorganizations, and cell spreading were inhibited in Chinese hamster ovary cells expressing full-length GP Ibalpha compared with GP Ibalpha lacking the 14-3-3 zeta binding site. Activation of Rho GTPases and cytoskeletal reorganizations were restored by expression of 14-3-3 zeta. Spreading in cells expressing truncated GP Ibalpha was inhibited by co-expressing a chimeric receptor containing interleukin 2 receptor alpha and GP Ibalpha cytoplasmic domain. These results identify a previously unrecognized function of 14-3-3 zeta, that of mediating integrin-induced signaling. They show that 14-3-3 zeta mediates Cdc42 and Rac activation. They also reveal a novel function of platelet GP Ib-IX, that of regulating integrin-induced cytoskeletal reorganizations by sequestering 14-3-3 zeta. Signaling across integrins initiates changes in cell behavior such as spreading, migration, differentiation, apoptosis, or cell division. Thus, introduction of the 14-3-3 zeta binding domain of GP Ibalpha into target cells might provide a method for regulating integrin-induced pathways in a variety of pathological conditions.

14-3-3 Proteins↗

PubMed2006

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide.

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli and is aberrantly elevated in a variety of human cancers. Rhabdomyosarcoma tumors are the most common soft-tissue sarcoma in childhood. In this investigation, we demonstrate that CK2 is a key survival factor that protects tumor cells from TRAIL-induced apoptosis. We have demonstrated that inhibition of CK2 phosphorylation events by 5,6-dichlorobenzimidazole (DRB) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis. CK2 inhibition also induced rapid cleavage of caspase-8, -9, and -3, as well as the caspase substrate poly(ADP-ribose) polymerase after TRAIL treatment. Overexpression of Bcl-2 protected cells from TRAIL-induced apoptosis in the presence of the CK2 inhibitor. Death signaling by TRAIL in these cells was Fas-associated death domain and caspase dependent because dominant negative Fas-associated death domain or the cowpox interleukin 1beta-converting enzyme inhibitor protein cytokine response modifier A prevented apoptosis in the presence of DRB. Analysis of death-inducing signaling complex (DISC) formation demonstrated that inhibition of CK2 by DRB increased the level of recruitment of procaspase-8 to the DISC and enhanced caspase-8-mediated cleavage of Bid, thereby increasing the release of the proapoptotic factors cytochrome c, HtrA2/Omi, Smac/DIABLO, and apoptosis inducing factor (AIF) from the mitochondria, with subsequent degradation of X-linked inhibitor of apoptosis protein (XIAP). To further interfere with CK2 function, JR1 and Rh30 cells were transfected with either short hairpin RNA targeted to CK2alpha or kinase-inactive CK2alpha (K68M) or CK2alpha' (K69M). Data show that the CK2 kinase activity was abrogated and that TRAIL sensitivity in both cell lines was increased. Silencing of CK2alpha expression with short hairpin RNA was also associated with degradation of XIAP. These findings suggest that CK2 regulates TRAIL signaling in rhabdomyosarcoma by modulating TRAIL-induced DISC formation and XIAP expression.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling.

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its beta-subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin alpha(IIb)beta(3) is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on alpha(IIb)beta(3), we examined alpha(IIb)beta(3)/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that alpha-actinin, myosin heavy chain, and Syk coimmunoprecipitate with alpha(IIb)beta(3) in resting platelets and that 120 dyn/cm(2) shear stress leads to their disassociation from alpha(IIb)beta(3). Shear-induced disassociation of alpha-actinin and myosin heavy chain from the beta(3) tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to alpha(IIb)beta(3). Syk's disassociation from beta(3) is inhibited when VWF binding to either GpIb-IX-V or alpha(IIb)beta(3) is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to alpha(IIb)beta(3) but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing alpha(IIb)beta(3) with beta(3) truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates alpha(IIb)beta(3) function and suggest that shear-induced alpha(IIb)beta(3)-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the beta(3)-tail.

Actinin↗

PubMed2001

Thyroid hormone receptor-interacting protein 1 modulates cytokine and nuclear hormone signaling in erythroid cells.

Erythropoietin (Epo) and thyroid hormone (T(3)) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T(3) receptor, providing a link between the Epo and T(3) signaling pathways. Trip-1 co-localized with Lyn and the T(3) receptor alpha in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T(3) stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T(3) reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27(Kip1) and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T(3)-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27(Kip1), and blocking T(3) response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2002

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) up-regulates death receptor 5 (DR5) mediated by NFkappaB activation in epithelial derived cell lines.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L) activates nuclear factor kappaB (NFkappaB). This activation is regulated by the recruitment of an adaptor protein Fas associating death domain (FADD) to TRAIL death receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5 TRAIL-R2). This leads to recruitment of caspase 8 and receptor interacting protein (RIP) to the receptor complex. Upon recruitment of caspase 8 and RIP, NFkappaB inducing kinase (NIK) becomes activated causing NFkappaB activation. The role of TRAIL induced NFkappaB activation in epithelial cells is unknown. Herein we demonstrate that TRAIL increases expression of DR5 in human embryonic kidney (HEK) 293, MCF-7 and MDA MB 231 epithelial cell lines while DR4 expression remains unchanged. Blockage of NFkappaB activation either by expression of dominant negative IkappaB or treatment with proteasome inhibitor lactacystin eliminates TRAIL induced DR5 expression. Expression of FADD dominant negative in HEK 293 cells that prevents the recruitment of caspase 8 and RIP to TRAIL death receptors also eliminates this increase. By over expression of the p65 subunit of NFkappaB that increases NFkappaB transcriptional activity, DR5 expression was increased compared to vector alone expressing cells. By blocking TRAIL induced NFkappaB activation, the sensitivity of cells to undergo TRAIL induced apoptosis was significantly decreased. Conversely, the amount of TRAIL induced apoptosis was increased in HEK 293 cells over expressing p65 subunit of NFkappaB. Finally blockage of NFkappaB activation eliminates the synergistic apoptotic response of TRAIL and etoposide. Thus, TRAIL mediated NFkappaB activation increases DR5 expression thereby amplifying the apoptotic response of TRAIL in epithelial derived cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Domain organization of D-AKAP2 revealed by enhanced deuterium exchange-mass spectrometry (DXMS).

Dual specific A-kinase anchoring protein 2 (D-AKAP2) is a scaffold protein that coordinates cAMP-mediated signaling complexes by binding to type I and type II protein kinase A (PKA). While information is unfolding regarding specific binding motifs, very little is known about the overall structure and dynamics of these scaffold proteins. We have used deuterium exchange-mass spectrometry (DXMS) and limited proteolysis to probe the folded regions of D-AKAP2, providing for the first time insight into the intra-domain dynamics of a scaffold protein. Deuterium on-exchange revealed two regions of low deuterium exchange that were surrounded by regions of high exchange, suggestive of two distinctly folded regions, flanked by disordered or solvent accessible regions. Similar folded regions were detected by limited proteolysis. The first folded region contained a putative regulator of G-protein signaling (RGS) domain. A structural model of the RGS domain revealed that the more deuterated regions mapped onto loops and turns, whereas less deuterated regions mapped onto alpha-helices, consistent with this region folding into an RGS domain. The second folded region contained a highly protected PKA binding site and a more solvent-accessible PDZ binding motif, which may serve as a potential targeting domain for D-AKAP2. DXMS has verified the multi-domain architecture of D-AKAP2 implied by sequence homology and has provided unique insight into the accessibility of the PKA binding site.

A Kinase Anchor Proteins↗

PubMed2004

Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression of invasiveness.

We recently established the critical role of the lipid phosphatase activity of the PTEN tumor suppressor in stabilizing cell-cell contacts and suppressing invasiveness. To delineate the effector systems involved, we investigated the interaction of PTEN with E-cadherin junctional complexes in kidney and colonic epithelial cell lines. PTEN and the p85 regulatory subunit of phosphatidylinositol 3-OH kinase (PI3K) co-immunoprecipitated with E-cadherin and catenins. By using a yeast two-hybrid assay, we demonstrated that PTEN interacted indirectly with beta-catenin by binding the scaffolding protein MAGI-1b. This model was corroborated in various ways in mammalian cells. Ectopic expression of MAGI-1b potentiated the interaction of PTEN with junctional complexes, promoted E-cadherin-dependent cell-cell aggregation, and reverted the Src-induced invasiveness of kidney MDCKts-src cells. In this model, MAGI-1b slightly decreased the activity of AKT, a downstream effector of PI3K. By using dominant-negative and constitutively active AKT expression vectors, we demonstrated that this kinase was included in the pathways involved in Src-induced destabilization of junctional complexes and was necessary and sufficient to trigger invasiveness. We propose that the recruitment of PTEN at adherens junctions by MAGI-1b and the local down-regulation of phosphatidylinositol-3,4,5-trisphosphate pools and downstream effector systems at the site of cell-cell contacts are focal points for restraining both disruption of junctional complexes and induction of tumor cell invasion.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗