Previdi Sara
Colombo Shana Maria
INTRODUCTION
p130Cas is an adaptor molecule that can act as scaffold for numerous partners. In addition it has an important role in multiple processes due to its capacity to regulate diverse signaling pathways (growth factor receptor tyrosine kinases, non-receptor tyrosine kinases, and integrins). p130Cas regulates also many cellular processes, including survival, proliferation, migration and apoptosis.Recent studies show that p130Cas is essential during early embryogenesis, with a critical role in cardiovascular development and it has been reported to be involved in the development and progression of several human cancers.
CAS-FAMILY
p130Cas is a member of the Cas family (Crk-associated substrate) of adaptor proteins which is characterized by the presence of multiple conserved motifs for protein–protein interactions, and by extensive tyrosine and serine phosphorylations. The Cas family contains other three proteins: NEDD9 (neural precursor cell expressed, developmentally down-regulated 9 also called human enhancer of filamentation 1, HEF-1), EFS (embryonal Fyn-associated substrate), and CASS4 (Cas scaffolding protein family member 4). These three Cas family proteins have high structural homology, conserved binding modules and effector proteins but their temporal expression, tissue distribution and functional roles are distinct. HEF1 is studied in epithelial and nervous tissues, where it regulates mitosis and neurite outgrowth, whereas EFS functions in T lymphocytes.
GENE
Breast Cancer Anti-estrogen Resistance 1(BCAR1), the official human gene name of p130Cas, is localized on chromosome 16 on region q and it is on the negative strand and has seven exons. There are 9 different isoforms that share the same sequence starting from the second exon to the last, but characterize by different starting sites. The longest isoform has 916 aa, the other isoforms use an alternative first exon.
Physical map of the human BCAR1 locus.
Sequence alignment of BCAR1 revealed extensive homology (91%) with rat and mouse p130Cas protein.
Bcar1 alignment with p130Cas. A Blast (www.ncbi.nlm.gov/blast) search with BCAR1 complementary DNA sequences in available databases demonstrated homology to rat and murine p130Cas sequences. An SH3 homology domain (boxed), a proline-rich sequence region (bold and underlined), and multiple potential tyrosine phosphorylation sites (shaded) are indicated.
STRUCTURAL FEATURES
p130Cas possesses an amino (N)-terminal Src-homology 3 domain (SH3) domain, followed by a proline-rich domain and a substrate domain. The substrate domain is comprised of 15 repeats of the YxxP consensus phosphorylation motif for Src family kinases (SFKs). Src-homology 2 (SH2)-domain containing proteins bind phospho-tyrosine residues in this region, recognizing their specific YxxP sequence as determined by the three amino acids following the phospho-tyrosine. Following the substrate domain is the serine-rich domain, which forms a four-helix bundle. This acts as a protein-interaction motif, similar to those found in other adhesion-related proteins such as focal adhesion kinase (FAK) and vinculin. The remaining carboxy-terminal sequence contains a bipartite Src-binding domain (residues 681–713) able to bind both the SH2 and SH3 domains of Src.
AMMINOACIDS SEQUENCE AND PERCENTUAGE
Human p130Cas aminoacid sequence. Highlighted are the sequences producing the Src homology 3 (SH3) domain, proline-rich domain, substrate domain, serine-rich domain, carboxy-terminal domain, and Src-binding domain. The 15 characteristic YxxP motifs are apparent within the substrate domain.
TYROSINE AND SERINE/THREONINE PHOSPHORYLATION
Tyrosine phosphorylation is the major post-translational modification of p130Cas, and occurs predominantly in the 15 YxxP repeats within the substrate domain. Tyrosine phosphorylation occurs as a result of a diverse range of extracellular stimuli, including growth factor stimulation (such as VEGF, PDGF, EGF, IGF, FGF2, MCSF), integrin activation, and vasoactive hormones and peptides ligands for G-protein coupled receptors (including endothelin, angiotensin and vasopressin).
During mitosis, when cells become rounder and temporarily detach from the extracellular matrix until cytokinesis is complete, p130Cas is phosphorylated on serine and threonine residues. This event determine the dissociation of a core focal adhesion complex composed of p130Cas, FAK, and Src, which occurs to allow cell detachment to progress. At re-entry into G1, serine/threonine phosphorylation is lost, probably through a PP2A.
SITE OF EXPRESSION AND SUBCELLULAR LOCALISATION
p130Cas is ubiquitously expressed and whose delection in mice is embryonic lethal.
Unphosphorylated p130Cas has a cytosolic distribution, whereas ligand-induced tyrosine phosphorylation promotes translocation of p130Cas to the cell membrane. p130Cas has been found to colocalise in focal adhesions with various focal adhesion associated proteins, including the kinases Src, FAK, PYK2, and the adaptor proteins Crk and Nck .The SH3 and carboxy-terminal domains of p130Cas are required for the localisation of p130Cas to focal adhesions. The group of Paola Defilippi discovered that the protein p140Cap is associated with p130Cas and actin cytoskeletal structures.
p130CAS IN HEALTH AND DISEASE
(p130Cas: a versatile scaffold in signaling networks,2006)
p130Cas in cell migration
The involvement of p130Cas in cell migration depends on its tyrosine phosphorylation by Src and on the assembly of a p130Cas–Crk–DOCK180 scaffold at adhesion sites (Temporal dissection of b1-integrin signaling indicates a role for p130Cas-Crk in filopodia formation, 2004). Scaffold formation drives localized Rac activation, leading to actin polymerization and the recruitment of high affinity integrin receptors that are necessary for lamellipodia extension and cell migration. p130Cas-null cells show defects in stress fiber formation, cell spreading, impaired actin bundling and cell migration (p130 (Cas), an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts, 1999), which are restored by the addition of full-length p130Cas. Several proteins negatively regulate the migratory functions of p130Cas. In STAT3-null keratinocytes, p130Cas is hyperphosphorylated on tyrosine residues and cells show increased adhesiveness and fast spreading on a collagen matrix (STAT3 deficiency in keratinocytes leads to compromised cell migration through hyperphosphorylation of p130(cas), 2002). This suggests that STAT3 negatively modulates p130Cas phosphorylation and cell migration by regulating the expression of molecules involved in aberrant p130Cas phosphorylation or by directly functioning as an adaptor molecule that recruits kinases or phosphatases to p130Cas. Finally, phosphatases might act on p130Cas itself. Cells expressing the activated phosphatase PTP-PEST exhibit a decrease in levels of tyrosine-phosphorylated p130Cas in parallel with defective migration.
p130Cas in survival and apoptosis
p130Cas is an important transducer of survival signals. Pro-survival signals emanating from the ECM and soluble growth factors and hormones proceed through their respective receptors, then through FAK and Src, to p130Cas, activating the small GTPases Ras and Rac, as well as JNK and Erk1/2–MAPK (Positional control of cell fate through joint integrin/receptor protein kinase signaling, 2003;). p130Cas is also required for integrin-dependent EGF-receptor activation, which in turn leads to cell survival.
p130Cas might also have a direct role in death signaling. Multiple pro-apoptotic stimuli, such as detachment from the ECM (known as anoikis), treatment with anticancer drugs or UVirradiation, induce p130Cas cleavage by caspase-3 or other proteases (Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells, 2002), generating a C-terminal 31-kDa fragment. This fragment promotes the loss of focal adhesions, cell rounding, nuclear condensation and fragmentation, which can be partially rescued by an uncleavable p130Cas mutant. The 31-kDa fragment contains a helix–loop–helix (HLH) domain that can heterodimerize with the transcription factor E2A and then translocate to the cell nucleus, where it contributes to cell death by physically preventing E-box binding by E2A. Moreover, the heterodimer inhibits E2A mediated p21 transcription, thus promoting cell death.
p130Cas in cardiovascular system
A critical role for p130Cas in cardiovascular development was first discovered with the generation of p130Cas knockout mice (Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas, 1998). Mice lacking p130Cas were growth retarded and died in utero at 11.5-12.5 embryonic days with poorly developed hearts exhibiting thin myocardium and dilated blood vessels, consistent with the observation that at this stage of development, p130Cas is predominantly expressed in the heart and vasculature of wild type mice.
p130Cas in cancer
p130Cas is emerging as an important player in the transformation and invasion driven by different oncogenes. Its involvement in c-Src-mediated tumorigenesis has been demonstrated by the inability of c-Src to transform p130Cas-null MEFs (Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas, 1998). It has also been reported recently that knockdown of p130Cas causes proliferative arrest in breast cancer cell lines harboring oncogenic mutations in KRAS, BRAF, PTEN, and PIK3CA (Ras- and PI3K-dependent breast tumorigenesis in mice and humans requires focal adhesion kinase signaling, 2009), underlying a role for p130Cas as a general regulator of breast cancer cell growth induced by different oncogenes. In particular, as its name indicated, the overexpression of BCAR1/p130CAS confers anti-estrogen resistance and the ability to proliferate in the presence of tamoxifen (BCAR1, a Human Homologue of the Adapter Protein p130Cas, and Antiestrogen Resistance in Breast Cancer Cells, 2000).
Summarises some of the functional roles of p130Cas in different types of human cancer.
CONCLUSION AND DIAGNOSTIC USE
Despite the increasing evidence showing the involvement of p130Cas in several human cancers, the in vivo relevance of p130Cas in tumorigenesis is not yet clear. p130Cas is not an oncogene per se but it is required for Src transformation, suggesting that p130Cas is an essential cofactor in the onset of cancer.
There is increasing interest in p130Cas as a potential therapeutic target, notably for the treatment of ovarian, breast and prostate cancer.There are however considerable challenges to overcome in the development of p130Cas targeted therapies. p130Cas is an adaptor molecule and lacks its own catalytic activity, making it more difficult to produce specific inhibitors.
Further work concerning the systematic mapping of phosphorylated residues and their relation to specific stimuli or functions may aid the search for competitors and inhibitors, and methods of inhibiting specific protein–protein interactions. Another approach would be to target particular downstream effectors that are implicated in tumour invasion and motility. p130Cas expression and phosphorylation levels may have use as a prognostic marker in some cancers, potentially assisting treatment and the prediction of drug effectiveness.
