Organization of functional domains in the docking protein p130Cas

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.     

Molecular basis for regulation of Src by the docking protein p130Cas

The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form.     

SH2-containing inositol 5'-phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates cellular adhesion and spreading

In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605-18609, 1998). We describe here a novel interaction between SHIP2 and the p130(Cas) adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130(Cas) association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130(Cas). The faster-migrating form of p130(Cas) was no longer recognized by antibodies to the amino terminus of p130(Cas) and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130(Cas) was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (DeltaRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.     

CrkI and p130(Cas) complex regulates the migration and invasion of prostate cancer cells

Prostate cancer metastasis is often associated with poor prognosis. The molecular coupling of the adaptor protein Crk to the docking protein p130(Cas) serves as a switch that regulates cell migration in several invasive cancer cells and Ack appears to act upstream of CrkII to modulate the cell motility. However, the precise role of Ack, Crk and p130(Cas) complex in prostate cancer migration remains unknown. In this study we examined the expression of Crk and p130(Cas) in prostate cancer cell lines, and found that CrkI and p130(Cas) protein level was higher in highly invasive PC-3M and PC-3 cell lines than in moderately invasive DU-145 cells. Upon shRNA mediated knockdown of CrkI and p130(Cas) in PC-3M cells, cell migration and invasion were significantly inhibited as analyzed by wound healing assay and transwell invasion assay. Furthermore, co-immunoprecipitation assay showed that p130(Cas) interacted with CrkI in PC-3M cells and the stability of p130(Cas) and CrkI depended on each other. AckI interacted with both CrkI and p130(Cas) and the interaction of AckI with CrkI seemed to be independent of p130(Cas) . Taken together, our results demonstrate the high expression of CrkI and p130(Cas) in invasive prostate cancer cells and the important role of CrkI/p130(Cas) complex in the migration and invasion of prostate cancer cells. These data suggest that CrkI/p130(Cas) could be exploited as potential molecular therapeutic target for prostate cancer metastasis.     

Identification of two focal adhesion targeting sequences in the adapter molecule p130(Cas)

The adapter molecule CAS is localized primarily within focal adhesions in fibroblasts. Because many of the cellular functions attributed to CAS are likely to be dependent on its presence in focal adhesions, this study was undertaken to identify regions of the protein that are involved in its localization. The SH3 domain of CAS, when expressed in isolation from the rest of the protein, was able to target to focal adhesions, whereas a variant containing a point mutation that rendered the SH3 domain unable to associate with FAK remained cytoplasmic. However, in the context of full-length CAS, this mutation did not prevent CAS localization to focal adhesions. Two other variants of CAS that contained deletions of either the SH3 domain alone, or the SH3 domain together with an adjoining proline-rich region, also retained the capacity to localize to focal adhesions. A second focal adhesion targeting region was mapped to the extreme carboxy terminus of CAS. The identification of this second focal adhesion targeting domain in CAS ascribes a previously unknown function to the highly conserved C terminus of CAS. The regulated targeting of CAS to focal adhesions by two independent domains may reflect the important role of CAS within this subcellular compartment.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.     

The phospho-docking protein 14-3-3 regulates microtubule-associated proteins in oocytes including the chromosomal passenger Borealin

Global regulation of spindle-associated proteins is crucial in oocytes due to the absence of centrosomes and their very large cytoplasmic volume, but little is known about how this is achieved beyond involvement of the Ran-importin pathway. We previously uncovered a novel regulatory mechanism in Drosophila oocytes, in which the phospho-docking protein 14-3-3 suppresses microtubule binding of Kinesin-14/Ncd away from chromosomes. Here we report systematic identification of microtubule-associated proteins regulated by 14-3-3 from Drosophila oocytes. Proteins from ovary extract were co-sedimented with microtubules in the presence or absence of a 14-3-3 inhibitor. Through quantitative mass-spectrometry, we identified proteins or complexes whose ability to bind microtubules is suppressed by 14-3-3, including the chromosomal passenger complex (CPC), the centralspindlin complex and Kinesin-14/Ncd. We showed that 14-3-3 binds to the disordered region of Borealin, and this binding is regulated differentially by two phosphorylations on Borealin. Mutations at these two phospho-sites compromised normal Borealin localisation and centromere bi-orientation in oocytes, showing that phospho-regulation of 14-3-3 binding is important for Borealin localisation and function.     

Phosphorylation-dependent interaction between plant plasma membrane H(+)-ATPase and 14-3-3 proteins

The H(+)-ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H(+)-ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H(+)-ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H(+)-ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H(+)-ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H(+)-ATPase and 14-3-3 proteins was identified and partially purified from maize root.     

Cyclin-dependent kinase 11p110 and casein kinase 2 (CK2) inhibit the interaction between tyrosine hydroxylase and 14-3-3

Tyrosine hydroxylase (TH) is regulated by the reversible phosphorylation of serines 8, 19, 31 and 40. Upon initiation of this study, serine 19 was unique due to its requirement of 14-3-3 binding after phosphorylation for optimal enzyme activity, although it has been more recently demonstrated that phosphorylated serine 40 also binds 14-3-3. To identify proteins that interact with TH following phosphorylation of serine 19, this amino acid was mutated to alanine and THS19A was used as bait in a yeast two-hybrid system. From this, mouse-derived cyclin-dependent kinase 11 (CDK11)p110 was identified as an interacting partner with THS19A. The interaction was confirmed using human CDK11p110 cDNA in a mammalian system. Previous research has demonstrated that casein kinase 2 (CK2) interacts with CDK11p110, and both were observed to phosphorylate TH in vitro. In addition, CDK11p110 overexpression was observed to inhibit the interaction between TH and 14-3-3. A mechanism contributing to disruption of the interaction between TH and 14-3-3 may be due to CK2 phosphorylation of specific 14-3-3 isoforms, i.e. 14-3-3 tau. Collectively, these results imply that CDK11p110 and CK2 negatively regulate TH catecholamine biosynthetic activity since phosphoserine 19 of TH requires 14-3-3 binding for optimal enzyme activity and a decreased rate of dephosphorylation.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast

The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.     

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.     

AND-34/BCAR3 regulates adhesion-dependent p130Cas serine phosphorylation and breast cancer cell growth pattern

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.     

Disruption of target cell adhesion structures by the Yersinia effector YopH requires interaction with the substrate domain of p130Cas

The docking protein p130Cas has, together with FAK, been found as a target of the Yersinia virulence effector YopH. YopH is a protein tyrosine phosphatase that is delivered into host cells via the bacterial type III secretion machinery, and the outcome of its activity is inhibition of host cell phagocytosis. In the present study using p130Cas-/- cells, and p130Cas-/- cells expressing variants of GFPp130Cas, we show that this docking protein, via its substrate domain, is responsible for subcellular targeting of YopH in eukaryotic cells. Since YopH inhibits phagocytosis, p130Cas was expected to be critical for signalling mediating bacterial internalization. However, p130Cas-/- cells did not exhibit reduced capacity to internalize Yersinia. On the other hand, when a dominant negative variant of p130Cas was expressed in these cells, the phagocytic capacity was severely impaired. Moreover, the p130Cas-/- cells displayed a marked reduced sensitivity towards YopH-mediated detachment compared to wild-type cells. Transfecting these cells with full-length p130Cas rendered cells hypersensitive to both mechanical and Yersinia-mediated detachment. This hypersensitivity was not seen upon transfection with the dominant negative substrate domain-deleted variant of p130Cas. This implicates p130Cas as a prominent regulator of cell adhesion, where its substrate-binding domain has a significant function.     

The interaction between ADAM 22 and 14-3-3zeta: regulation of cell adhesion and spreading

The ADAM family consists of a number of transmembrane proteins that contain disintegrin-like and metalloproteinase-like domains. Therefore, ADAMs potentially have cell adhesion and protease activities. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in the regulation of various cellular functions. Here we report the identification of a novel interaction between the ADAM 22 cytoplasmic tail and the 14-3-3zeta isoform by a yeast two-hybrid screen. The interaction between the ADAM 22 cytoplasmic tail and 14-3-3zeta was confirmed by an in vitro protein pull-down assay as well as by co-immunoprecipitation, and the binding sites were mapped to the 28 amino acid residues of the C-terminus of the ADAM 22 cytoplasmic tail. Furthermore, we found that overexpression of the ADAM 22 cytoplasmic tail in human SGH44 cells inhibited cell adhesion and spreading and that deletion or mutation of the binding site for 14-3-3zeta within the ADAM 22 cytoplasmic tail abolished the ability of the overexpressed cytoplasmic tail to alter cell adhesion and spreading. Taken together, these results for the first time demonstrate an association between ADAM 22 and a 14-3-3 protein and suggest a potential role for the 14-3-3zeta/ADAM 22 association in the regulation of cell adhesion and related signaling events.     

Phosphorylation of grb10 regulates its interaction with 14-3-3

Grb10 is a member of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. Grb10 expression levels can influence Akt activity, and Grb10 may act as an adapter involved in the relocalization of Akt to the cell membrane. Here we identified 14-3-3 as a binding partner of Grb10 by employing a yeast two-hybrid screen. The 14-3-3.Grb10 interaction requires phosphorylation of Grb10, and only the phosphorylated form of Grb10 co-immunoprecipitates with endogenous 14-3-3. We could identify a putative phosphorylation site in Grb10, which is located in a classical 14-3-3 binding motif, RSVSEN. Mutation of this site in Grb10 diminished binding to 14-3-3. Thus, Grb10 exists in two different states of phosphorylation and complexes with 14-3-3 when phosphorylated on serine 428. We provide evidence that Akt directly binds Grb10 and is able to phosphorylate Grb10 in an in vitro kinase assay. Based on these findings, we propose a regulatory circuitry involving a phosphorylation-regulated complex formation of Grb10 with 14-3-3 and Akt.     

14-3-3 proteins in membrane protein transport

14-3-3 proteins affect the cell surface expression of several unrelated cargo membrane proteins, e.g., MHC II invariant chain, the two-pore potassium channels KCNK3 and KCNK9, and a number of different reporter proteins exposing Arg-based endoplasmic reticulum localization signals in mammalian and yeast cells. These multimeric membrane proteins have a common feature in that they all expose coatomer protein complex I (COPI)- and 14-3-3-binding motifs. 14-3-3 binding depends on phosphorylation of the membrane protein in some and on multimerization of the membrane protein in other cases. Evidence from mutant proteins that are unable to interact with either COPI or 14-3-3 and from yeast cells with an altered 14-3-3 content suggests that 14-3-3 proteins affect forward transport in the secretory pathway. Mechanistically, this could be explained by clamping, masking, or scaffolding. In the clamping mechanism, 14-3-3 binding alters the conformation of the signal-exposing tail of the membrane protein, whereas masking or scaffolding would abolish or allow the interaction of the membrane protein with other proteins or complexes. Interaction partners identified as putative 14-3-3 binding partners in affinity purification approaches constitute a pool of candidate proteins for downstream effectors, such as coat components, coat recruitment GTPases, Rab GTPases, GTPase-activating proteins (GAPs), guanine-nucleotide exchange factors (GEFs) and motor proteins.