Structure and Binding Partners

Cortactin is expressed in all tissues except cells of myeloid lineage, where it is functionally replaced by the related protein HS1. Based on primary sequence analysis, cortactin is subdivided into several distinct domains (Fig. 1). The amino-terminal domain (NTA) contains a series of acidic residues and a binding motif that interacts with the Arp2/3 complex. The NTA domain is followed by a series of 37 amino acid tandem repeats, six complete and one incomplete in the predominant isoform. The repeat region interacts with F-actin, with binding activity centered around the fourth repeat. Alternative splicing in some cells is responsible for two additional isoforms that lack either the sixth complete or fifth and sixth complete repeat segments. These forms bind F-actin at reduced affinities. Following the repeats region is an alpha-helical domain that is the site of cleavage by the protease  calpain 2. This is followed by a proline-rich region that harbors serine, threonine, and tyrosine residues that serve as the primary sites of phosphorylation. An SH3 domain is found at the extreme carboxyl terminus that binds to proline-rich sequences on a variety of proteins including the actin regulatory proteins N-WASp, WASp-interacting protein and the missing in metastasis protein, the endocytic proteins dynamin 2 and CD2AP, the small  GTPase regulatory proteins FGD1 and AMAP1, scaffolding proteins of the SHANK family, and the  tight junction protein ZO-1. These structural parameters and binding partners allow cortactin to function as a molecular scaffold by linking a wide variety of diverse regulatory molecules to sites of Arp2/3-mediated actin assembly.

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Function

The function of cortactin has been best defined in regard to cell motility. Downregulation of cortactin protein expression reduces cellular movement, while overexpression of cortactin enhances this process. Biochemical studies have determined that cortactin activates Arp2/3 complex actin nucleation activity through the NTA domain, and its localization within lamellipodia indicates that cortactin contributes to the formation of the dendritic cortical actin network responsible for lamellipodia protrusion. Important in this aspect is the ability of cortactin to stabilize Arp2/3-produced actin networks, a feature unique among Arp2/3-activating proteins that serves to prolong the half-life of branched F-actin filaments at the cell periphery. Accordingly, cortactin depletion reduces the ability of extended lamellipodia to persist and inhibits efficient leading edge dynamics. Cortactin can effect Arp2/3-mediated actin polymerization by additional alternative mechanisms, most notably by activation of the Arp2/3 regulatory protein N-WASp through association with the cortactin SH3 domain. Cortactin fragments lacking the NTA but containing the SH3 domain are capable of stimulating motility, suggesting that the NTA and SH3 domains can function independently with regard to promoting actin-based cell movement. The interaction of cortactin with dynamin 2 is also noteworthy in that cortactin is recruited to subpopulations of clathrin-coated pits by dynamin 2 and is important for driving the scission of invaginating pits to produce intracellular endocytic vesicles. The cortactin-dynamin complex is also important in regulating cell morphology, invadopodia function, and the genesis of vesicles from the trans-Golgi network.

Regulation

Evidence to date indicates that phosphorylation on tyrosine and serine residues is the main factor involved in regulating cortactin function, although the precise mechanisms are unclear. Activation of  receptor tyrosine kinases or adhesion molecules leads to phosphorylation of three tyrosine sites in the proline-rich domain that are required for efficient cell migration. These sites are direct targets of  Src and related non-receptor tyrosine kinases and are hyperphosphorylated by oncogenic variants (i.e., v-Src). Tyrosine-phosphorylated cortactin is enriched within lamellipodia and invadopodia, indicating a potential role in regulating cortical actin dynamics and has been shown to influence F-actin architecture. Cortactin is also phosphorylated on two serine residues by  MAP kinase in the proline-rich domain, and dual phosphorylation of cortactin by Src and MAP kinase has opposing effects on the ability of the cortactin SH3 domain to interact with and activate N-WASp. This has led to the proposal of a regulatory phosphorylation switch mechanism predicated by cortactin initially existing in an autoinhibited closed conformation, with the SH3 domain binding back and interacting with motifs in the proline-rich domain. Phosphorylation of cortactin by MAP kinase induces a conformation change that renders the SH3 domain accessible for binding and activating N-WASp, whereas phosphorylation of cortactin by Src causes disassociation of N-WASp from the SH3 domain and subsequent downregulation of N-WASp activity. This proposal remains theoretical in part since it is derived primarily from biochemical analysis and evidence for an intramolecular cortactin interaction is lacking. In addition to phosphorylation indirectly regulating N-WASp activity, the serine/threonine kinase PAK1 phosphorylates cortactin within the first tandem repeat, resulting in reduced F-actin binding. Subsequent work has identified over 17 additional phosphorylation sites in every domain except the SH3, but the responsible signaling pathways and functional significance of these modifications are currently unknown. Besides phosphorylation, cortactin is also regulated by the calcium-dependent protease calpain 2, which cleaves cortactin between the repeats and alpha-helical domain and is important in limiting the extent of lamellipodia protrusion.

Role in Cancer

The cortactin gene (CTTN, formerly EMS1) maps to chromosome 11q13.3, a region that is frequently amplified in a number of cancers with inherently high invasive and metastatic potential, including  Brms1, head and neck, ovarian, bladder, and  hepatocellular carcinomas. 11q13 and CTTN amplification is associated with poor pathological outcome parameters including increased tumor recurrence, advanced disease stage, poor histological differentiation, increased lymph node metastasis, and reduced disease-specific survival. Mechanistically, cortactin overexpression as a result of CTTN amplification increases tumor cell motility and invasion as well as preventing the internalization and ubiquitylation-mediated degradation of EGF receptor, a receptor tyrosine kinase often overexpressed in carcinomas that is a potent activator of Src and MAPK. Sustained EGF receptor activity as a result of CTTN amplification and cortactin overexpression promotes increased cortactin tyrosine phosphorylation, which has been shown to enhance distant metastasis of breast carcinoma cells in  mouse models. EGF receptor inhibitors suppress tumor cell invasion and cortactin tyrosine phosphorylation, providing further support for the clinical relevance of cortactin phosphorylation in human cancer. Specific functions for cortactin in tumor cell invasion have been identified, most notable being its absolute role in the signaling and structural requirements governing the formation and function of invadopodia (Fig. 2). Cortactin is required to recruit and sequester the main invadopodial  matrix metalloproteinase MT1-MMP into sites of newly initiated invadopodia. Cortactin tyrosine phosphorylation levels within invadopodia correlate to the degree of extracellular matrix degradation activity, but the functional significance of cortactin phosphorylation in invadopodia is currently undefined. Cortactin in invadopodia forms a complex with the focal adhesion protein  paxillin and other signaling proteins. Often present in invasive carcinomas is amplification and overexpression of AMAP1, which physically links paxillin and cortactin together in promoting tumor invasion. Targeting of the trimeric paxillin-AMAP1-cortactin complex with competitive peptides mimicking the AMAP1 binding site for the cortactin SH3 domain suppresses carcinoma invasion and may show potential value in antimetastatic therapy. In addition to its role in promoting tumor cell invasion and metastasis, cortactin has been shown to be a prominent tumor antigen and is present at high levels in the sera of subsets of breast cancer patients. Recent work has determined that cortactin is an extracellular ligand for TEM7, a transmembrane receptor expressed primarily on the surface of tumor endothelial cells. While the function of TEM7 is currently unknown, related TEM proteins promote endothelial cell growth and survival, raising the possibility that cortactin released into the circulation from necrotic or damaged tumor cells, especially tumors with CTTN amplification, may serve an unexpected role by promoting or maintaining tumor  angiogenesis.

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