DEFINIZIONE
P140-cap is a 140 kDa novel adaptor protein associated with P130-Cas and actin cytoskeletal structures; Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.
It’s a component of The Cap family consists of two members, p140CAP and sickle-tail (SKT), multisite docking proteins, which are characterized by conserved sequence motifs that can associate with multiple
Effectors.
The p140Cap protein is expressed in both human and mouse cells and tissues. Indeed, sequence comparison between human, mouse, rat, fish, and Drosophila genomes revealed the existence of ortholog
genes, coding for highly homologous proteins in all these species (www.ensembl.org).
The carboxy-terminal part of p140Cap contained a proline-rich region and a charged amino acid stretch, which by sequence comparison, was shown to be highly conserved among human, mouse, and fish species:
1. Multiple alignment of human (Hu), mouse (Mo), and fugu (Fu) amino acids of The carboxy-terminal region. *, single, fully conserved residues, :, conservation of strong groups; ., conservation of weak groups. Box: highly conserved proline-rich region
The interaction between p140Cap and
p130Cas is mediated by the last 217 amino acids of the p140Cap carboxy-terminal region. Conversely, p130Cas associates with p140Cap in the region encompassing amino
acids 544–678. It’s identified in the p140Cap carboxy-terminal region a charged amino acid-rich domain and a proline-rich region, putative binding sites for the SH3 domains of Abl, Crk, and Itk proteins.
Similarly, the region of p130Cas that associates with the carboxy-terminal fragment of p140Cap in the pulldown experiments is characterized by a proline-rich region that can bind to the SH3 domains of several transducing molecules and by the presence of a tyrosine in position 668 that mediates binding to the Src SH2 domain.
THE GENE
SRCIN1, wich encodes P140, is located on human chromosome 17q12 and its flanking regions contain several genes that are involved in tumour initiation and progression, such as ERbb2 (17q12), bRCA1 (17q21), retinoic acid receptor-α (RARA; 17q21) and signal transducer and activator of transcription 3 (STAT3; 17q21).
CHEMICAL STRUCTURE AND IMAGE
2. p140 Cas-associated protein (p140CAP; also known as SRCIN1) consists of an N terminal tyrosine-rich region (TYR RICH), an actin-binding domain (ABd), a proline-rich domain (Pro1), a coil-coiled region (C1–C2), two domains rich in charged amino acids (CH1 and CH2) and a C terminal proline-rich domain (Pro2). SRC, p130CAS, MAPRE3 (also known as EB3) and vinexin bind to the Pro2 domain of p140CAP.
Genomic organization of p140-cap
The molecule did not contain a signal sequence nor a transmembrane domain and presented a glycine-rich region, two proline-rich motifs, two coil-coiled domains, and two C-terminal charged amino acid-rich regions.
3. Amino acid sequence of p140Cap. Indicated are the predicted glycine-rich region (underlined in dark gray), the proline-rich domains (boxed), the putative SH3 binding regions (dashed black underlined), the coiled-coil domains (underlined in white), and the charged sequence (underlined in light gray).
AMMINOACIDS PERCENTUAGE
Blasting mouse p140Cap cDNA on the mouse genome allowed the mapping of a single p140 gene on chromosome 11 with a coding sequence spanning on 22 exons. However, blasting the first 163 bases of the mouse 5_-untranslated cDNA on the human genome revealed the presence of an pstream additional exon that we called 1a. Similarly, blasting the rat cDNA on the human genome showed that the 5_-untranslated region matched to another upstream exon (1b) located between 1a and 2.
Synthesis and turnover
P140 is highly expressed in brain, testis and ephitelial-rich tissues such as lung and mammary gland.
Cellular function and localization
To investigate the function of p140 in cells, they are used different cellular models:
- in ECV304 cells plated on fibronectin in the presence of serum, p140Cap clearly localizes on stress fibers that extend throughout the cell body and on cortical actin, indicating an association of p140Cap with actin-enriched structures.
the p140Cap is strictly connected to the actin cytoskeleton, suggesting a role in actin cytoskeleton remodeling. In addition to the colocalization with actin stress fibers, p140Cap seems to colocalize with p130Cas in a cytoplasmic punctuate staining as well as in specific membrane ruffle structures induced by PMA treatment. The association of p140Cap and p130Cas in the cytoplasm and in ruffles and the codistribution of p140Cap with actin cables strongly suggest that p140Cap plays an important role either in the structural organization of the actin cytoskeleton, which affects cell spreading on extracellular matrix, or in functional regulation of cell motility.
- p140Cap is tyrosine phosphorylated in response to adhesion or soluble mitogens such as
serum and EGF. In HEK293, EGF treatment induces a rapid tyrosine phosphorylation of transfected p140Cap, which is dependent on the kinase activity of the EGFR itself, as demonstrated
by using the specific EGFR inhibitor AG1478. These results demonstrate that p140Cap is a novel downstream effector of the EGFR in response to its ligand, opening a new perspective for analysis of possible functional roles of p140Cap.
Integrin-mediated adhesion induces strong phosphorylation of cellular proteins that have been shown to play multiple functions affecting cell polarity, migration, growth, and survival. Plating HEK293 cells on integrin ligands stimulates p140Cap phosphorylation within 15 min of cell adhesion, demonstrating that p140Cap tyrosine phosphorylation is also modulated by integrin- dependent adhesion.
p140Cap is tyrosine phosphorylated in response to adhesion, serum, or EGF treatment. (A) p140Cap-transfected HEK293 cells, serum-deprived for 24 h, were detached and plated for different times on dishes coated with mAb L230 against the _v integrin subunit or kept in suspension for 30 min. Cells were detergent extracted at the indicated times, and extracts were immunoprecipitated using antibodies to p140Cap. The immunoprecipitates were blotted with anti-phosphotyrosine antibody (top) and reblotted with antibodies to p140Cap (bottom). (B) Cells treated as described in A, kept in suspension or plated on FN for the indicated times, were extracted and immunoprecipitated using antibodies to phosphotyrosine (p-Tyr), and the immunoprecipitates were blotted with anti-p140Cap antibodies (top) and reblotted with antibodies to p130Cas (bottom). © Cells were treated with 20% FCS or with 50 ng/ml human recombinant EGF for 30 min. (D) Cells were treated with 50 ng/ml human recombinant EGF for 30 min in the presence of 250 nM AG1478. The p140Cap immunoprecipitates were blotted with anti-phosphotyrosine antibody (top) and reblotted with antibodies to p140Cap (bottom). The results are representative of three independent experiments
- p140Cap plays a relevant role in the early phases of cell adhesion inhibiting NIH3T3 and ECV304 cell spreading on fibronectin. In fact, when cells were plated on fibronectin, p140Cap expression dramatically affects actin cytoskeleton organization, maintaining the cells in a round shape. This effect was abolished when the mutant p140-ΔCT was expressed, indicating that the region involved in p130Cas association is required for this effect.
Overexpression of p140Cap affects cell spreading in NIH3T3 cells. (A) NIH3T3 cells were transfected with pEGFP-N2 (a–c), pEGFP-p140Cap (d–f), or pEGFP-p140-ΔCT cDNAs (g–i). Forty-eight hours after transfection,
cells were detached from culture dishes and plated for 1.5 h on fibronectin-coated glass coverslips. Cells were than fixed, permeabilized, and stained using rhodamine-labeled phalloidin (Phd).
FUNCTION IN TUMOR
The major function of the p140CAP adaptor in tumour cells is to regulate SRC kinase activation. In particular,on cell–ECM adhesion or epidermal growth factor (EGF) stimulation of breast cancer cells, p140CAP activates c-src tyrosine kinase (CSK), which causes the closure of SRC in an inactive conformation by phosphorylating the inhibitory tyrosine on the C terminal domain of SRC. Therefore, p140CAP represents a new potent regulator of the proto-oncogene SRC that is able to shift the balance from active to inactive SRC. Consequently, integrin signalling that is dependent on SRC, such as tyrosine phosphorylation of FAK, and p130CAS and RAC1 activation, is impaired in cells expressing high levels of p140CAP.
Using MCF7 as cellular model, it is demonstrated that p140Cap overexpression compromises integrin-dependent Src activation and downstream signaling:
- MCF7-Mock cells Src activity was upregulated within 30 min of adhesion to FN, Src activity was not induced in p140/P9 cells. Src kinase activity was also evaluated by phosphospecific antibodies against the critical tyrosine residue 416 in the Src kinase domain. Upon FN adhesion, tyrosine 416 was phosphorylated only in Mock cells, but not in p140Cap-overexpressing cells (Figure 4A, right panel). Taken together, these results demonstrate that high levels of p140Cap result in inhibition of integrin-dependent Src activation. Upon integrin-mediated adhesion, Src phosphorylates downstream effectors such as FAK and p130Cas. To investigate whether p140Cap overexpression affects this signalling, both MCF7-Mock and p140/P9 cells were analysed for phosphorylation of FAK. As depicted in Figure 4B (left panel), p140Cap overexpression did not affect integrin-dependent autophosphorylation on FAK tyrosine 397. In contrast, tyrosine 925, a residue shown to be a specific substrate of Src kinase, was not phosphorylated in p140/P9 cells (Figure 4B, right panel). Moreover, the binding between FAK and Src is decreased in p140/P9 cells comparedto Mock-transfected cells (Figure 4C). These results suggest that high levels of p140Cap do not modify integrin-dependentFAK autophosphorylation but rather, alter its ability to associate with Src, thereby affecting Src-dependent phosphorylation on specific residues. Moreover, integrin-dependent phosphorylation of p130Cas (Figure 4D) were decreased in cells overexpressing p140Cap.
- p140Cap-dependent Src kinase inhibition is mediated by Csk activation. Csk is a potent negative regulator of Src, due to its ability to phosphorylate the negative regulatory tyrosine 527 on the carboxy-terminal domain of Src. Infection with adenoviruses expressing a kinase-defective Csk mutant (Csk-KD) induced a strong activation of Src in MCF7-p140/P9 cells, indicating that the presence of the kinase-defective Csk counteracts the ability of p140Cap to inhibit Src activity. These data were further supported by the analysis of Src activity in MCF7-Mock and MCF7-p140/P9 cells transiently transfected with Csk siRNA. Reciprocal immunoprecipitation of p140Cap and Csk from MCF7-Mock cells showed that the two molecules co-immunoprecipitated, revealing the association of Csk and Src with p140Cap in a macromolecular complex, which could favour the regulation of kinase activities. Toinvestigate the direct interaction between p140Cap and Csk, we performed Far-Western analysis on p140Cap and GAPDH (negative control) immunoprecipitates using recombinant Csk produced by in vitro transcription/translation as probe. the Csk antibody detected a band at 140 kDa only in the p140Cap and not in the GAPDH immunoprecipitates, indicating that the Csk protein binds to p140Capon the filter. Therefore, this experiment demonstrates that the p140Cap and Csk directly interact.
In tumour cells p140CAP also regulates E-cadherin dependent cell–cell adhesion. p140CAP regulates cell–cell contact dynamics by increasing the amount of immobilized E-cadherin at the cell surface, thereby regulating the strength of cell–cell adhesion. This mechanism also depends on the inhibition of SRC kinase activity. p140CAP also regulates the Ras pathway through an additional unknown mechanism. Therefore, in cancer cells, p140CAP regulates EGFR signalling through a dual mechanism, which involves E-cadherin-dependent inactivation of EGFR and Ras-dependent inhibition of ERK1–ERK2–MAPK activity.Interestingly, p140Cap expression is lost in more aggressive human breast cancers, showing an inverse correlation with EGFR expression. Therefore, p140Cap mechanistically behaves as a tumour suppressor that inhibits signalling pathways leading to aggressive phenotypes.
Migration and invasion. p140CAP decreases the ability of breast cancer cells to spread on ECM proteins and
to migrate and invade in in vitro assays. Consistently, p140CAP silencing accelerates the early phases of cell
spreading on ECM, induces a fibroblastic-like morphology, and increases motility and invasion. In addition,
p140CAP specifically interferes with the ability of both breast and colon cancer cells to scatter from a compact colony in response to EGF. The mechanism by which p140CAP interferes with cell scattering is based on the ability of p140CAP to immobilize E-cadherin at the cell membrane, as described above.
Actin cytoskeleton remodelling is a crucial requirement for cell scattering and motility. p140CAP has been
shown to co-localize with actin stress fibres and cortical actin and to associate with proteins that are involved in actin cytoskeleton dynamics, such as p130CAS, SRC, vinexin and cortactin. These findings, along with the presence of a putative actin-binding domain in p140CAP, suggest that this adaptor could be both
directly and indirectly involved in actin filament assembly.
Proliferation. In addition to cell motility and invasion, the ability of p140CAP to regulate SRC and Ras pathways also profoundly affects cell proliferation. Increasedexpression of p140CAP in both breast and colon cancer cells inhibits proliferation in vitro, but does not affect cellsurvival. Interestingly, in breast cancer cells, p140CAP expression controls anchorage-independent growth, probably by inhibiting downstream integrin signalling, such as SRC and RAC1 activation. p140CAP might regulate ER signalling, contributing to breast cancer resistance to hormonal therapies. In conclusion, p140CAP functions as a tumour suppressor protein in breast and colon cancer cells, with a broad effect on cell proliferation and
tumour growth.
IN VIVO FUNCTION
p140Cap is expressed in rat brain in a developmental stage dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults.
Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons.
Localization of p140Cap in primary cultured rat hippocampal neurons
Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a presynaptic protein, synaptophysin, interacts with p140Cap.
p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling. In addition to the above functions in non-neuronal cells, p140Cap appears to be involved in yet unidentified cellular events in neurons as this protein was also identified as a synaptosome-associated protein of 25 kDa (SNAP-25)-interacting protein and termed SNIP. SNAP-25 is a component of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs), playing a major role in membrane docking of synaptic vesicles during neurotransmitter release. As subcellular fractionation analyses suggest that p140Cap interacts with the cortical actin cytoskeleton as well as SNAP-25, it appears to serve as a linker protein connecting SNAP-25 to the submembranous cytoskeleton. p140Cap is expressed in a developmental stage dependent manner in rat brain; the protein was detected at E14.5 and dramatically increased by E18.5. In adult rat brain, p140Cap appeared to be dominantly present in synapses, especially excitatory ones. p140Cap is relatively abundant in the synaptic plasma membrane. Electron microscopy
analyses then revealed that p140Cap is located mainly at post-synapses but also at pre-synapses. These results suggest that p140Cap plays roles in the formation and maintenance of synapses during brain developmental processes. p140Cap is implicated in neuronal secretion as it has been reported to interact with a SNARE protein, SNAP-25, in vitro p140Cap interacts with another synaptic vesicle protein, synaptophysin, which is not a component of the SNARE complex. Synaptophysin is reported to control the
targeting of synaptobrevin to synaptic vesicles. Thus, synaptophysin is most likely to serve as a regulator for
the incorporation of synaptobrevin into the SNARE complex. Given our results and the findings reported by Chin et al. p140Cap may take part in the regulation of neurotransmitter release at two protein (synaptophysin and SNAP-25) levels, although the physiologic significance and the precise regulatory mechanisms remain to be clarified. We here identified vinexin, a member of a multidomain adaptor family, as a binding partner for p140Cap. Like p140Cap, vinexin is enriched at synapses and is suggested to play as yet unidentified physiologic role(s) there. Although the physiologic significance of the vinexinp140Cap interaction at synapses is not known, the results obtained here suggest that subcellular localization of these
proteins is affected by each other, and that p140Cap alters mode of interaction of vinexin with other proteins. Vinexinp140Cap complex might be involved in the formation and/or maintenance of synapses. p140Cap may be involved in synapse functions, such as neurotransmitter release, signaling, and synapse formation/maintenance.
P140Cap is also a EB3 binding partner, binds to the Src kinase substrate and F-actin binding protein cortactin. Overexpression of p140Cap or cortactin rescues the EB3 depletion phenotype. The interactions of EB3-positive MT plus-ends, p140Cap and cortactin may therefore represent a link between the local signaling of MTs and the actin cytoskeleton within the dendritic spines.
DIAGNOSTIC USE
Regarding p140CAP and its potential role as a tumour suppressor, the generation of decoy proteins that could function as the intact protein, may have important potential therapeutic applications. Moreover, human breast cancer analysis shows that p140CAP expression is inversely correlated with the aggressiveness of malignancy, suggesting that p140CAP could be a new prognostic factor for the diagnosis of poorly aggressive breast tumours. Structure-based design of inhibitors should also be developed to target specific domains of these adaptor proteins, particularly regions that contain tyrosine or serine residues, or regions that are involved in binding with specific signalling effectors, such as PI3K, SRC, FAK and AKT.
