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Src family kinases (SFKs), non-receptor-type protein tyrosine kinases, are originally identified as the products of proto-oncogenes and are subsequently implicated in the regulation of cell proliferation and differentiation in the developing mammalian brain. These studies stem from work on the Rous sarcoma virus, a chicken tumor virus discovered in 1911 by Peyton Rous. V-Src (a viral protein) is encoded by the avian cancer-causing oncogene of Rous sarcoma virus. In contrast, Src (the normal cellular homologue) is encoded by a physiological gene, the first proto-oncogene to be described and characterized. Recent studies using transgenic mouse models have demonstrated that SFKs that are highly expressed in the adult brain regulate neuronal plasticity and behavior by tyrosine phosphorylation of key substrates such as neurotransmitter receptors.
Structure, function, and expression of SFKs
Cellular-Src (short for “sarcoma”) is the first proto-oncogene discovered and its protein product Src is the prototype of this family of PTKs. There are 8 members of the family, including Src, Fyn, Yes and Fgr, which make up the SrcA subfamily, and Lyn, Hck, Lck, and Blk, which make up the SrcB subfamily. The Src-related kinases Frk, Srm and Brk are referred to as SFKs, but they lack structural features common to all SFKs. Src, Fyn and Yes are widely expressed, but the remaining SFKs are primarily expressed in hematopoietic cells.
SFKs consist of an N-terminal myristoyl group attached to a Src homology 4 (SH4) domain, followed by a unique region, an SH2 domain, an SH3 domain, a proline-rich SH2-kinase linker region, and an SH1 or kinase domain. Myristoylation of the N-terminus facilitates binding to the inner leaflet of plasma membrane, which is required for function. The SH2 and SH3 domains of SFKs mediate intra- and intermolecular interactions with proline-rich regions and phosphotyrosine residues, respectively. In addition, all SFKs contain 2 highly conserved tyrosine residues, one in their C-terminal tail and the other in their activation loop that is critical for regulating SFK activity. Phosphorylation of the C-terminal tyrosine residue by C-terminal Src kinase (Csk) or its family member Csk homologous kinase inhibits SFK activity, whereas trans-autophosphorylation of the activation loop tyrosine residue maximally activates the SFK. Conversely, dephosphorylation of the C-terminal inhibitory and activation loop tyrosine residues by PTPs increases and decreases SFK activity, respectively (Figure 1).
Figure 1. The general structure of SFKs.
Roles of SFKs in the regulation of synaptic transmission
Recent studies have indicated that SFKs regulate neuronal plasticity and behavior through tyrosine phosphorylation of neurotransmitter receptors. These receptors include ionotropic glutamate receptors of the NMDA and AMPA subclasses (NMDARs and AMPARs), which contribute to excitatory transmission, as well as GABA type A receptors (GABAARs), which mediate the majority of fast synaptic inhibition in the adult mammalian brain.
NMDARs are ligand-gated cation channels that allow the flow of K+, Na+ and Ca2+ in response to the binding of glutamate. They are thought to be tetramers that consist of two GluN1 (formerly known as NR1) and two other modulatory subunits, comprised of either GluN2A-D (NR2A-D) or GluN3A-B (NR3A-B) subunits. A role for SFKs in the regulation of ligand-gated neurotransmitter receptor function was first indicated by the observation that the intracellular application of Src in hippocampal slices resulted in potentiation of NMDA-induced current. Treatment with an SFK-activating peptide (pYEEI) was also found to increase NMDAR currents in cultured neurons or hippocampal slices. In addition, activation of SFKs was shown to be important for the induction of long-term potentiation (LTP), a form of synaptic plasticity that is important for learning and memory, in CA1 pyramidal neurons of hippocampal slices.
Figure 2. Regulation of AMPAR endocytosis and function by SFKs.
GABAARs are Cl–selective ligand-gated ion channels. More than 20 distinct GABAAR subunits exist, but the majority of GABAARs in the adult CNS are composed of two α subunits, two β subunits, and one γ subunit (Figure 3). The γ2 and β1 subunits of GABAARs were first shown to be tyrosine-phosphorylated by v-Src, an active mutant of Src, in vitro and in A293 cells. The γ2 subunit of GABAARs has also been shown to be tyrosine-phosphorylated under basal conditions in cultured cortical neurons and in adult rat brain. Tyrosine phosphorylation of the γ2 subunit induced by vanadate, a broad-spectrum inhibitor of PTPs, was found to be markedly reduced in hippocampal slices of Fyn-deficient mice, suggesting that SFKs, in particular Fyn, are important for the tyrosine phosphorylation of this GABAAR subunit in the brain.
Figure 3. Regulation of GABAAR endocytosis and function by SFKs.
Src signaling and cancer
Src is a non-receptor protein tyrosine kinase that participates in numerous signaling pathways. Src interacts with several protein-tyrosine kinase receptors at the plasma membrane. The flow of information is bi-directional with the receptors affecting Src activity and vice versa. Src interacts with EGFR (ErbB1) and ErbB2, two key protein-tyrosine kinase receptors. EGFR mutations occur in NSCLC and ErbB2 overexpression is associated with breast cancer. The ErbB family is also implicated in colorectal, stomach, head and neck, and pancreatic carcinomas as well as glioblastoma. Besides protein-tyrosine kinases, Src-family kinases are controlled by integrin receptors, G-protein coupled receptors, antigen-and Fc-coupled receptors, cytokine receptors, and steroid hormone receptors. Src participates in cell migration and motility by interacting with integrins, E-cadherin, and focal adhesion kinase (Figure 4). Src participates in pathways regulating cell survival, proliferation, and regulation of gene expression. The enzyme also plays an essential role in bone formation and remodeling and may play a role in breast, prostate, and lung cancer metastasis to the skeleton.
Figure 4. Src signaling pathways.
Therapeutic small molecule Src inhibitors
The role of v-Src in oncogenesis eventually led to the discovery of the Src proto-oncogene and then to the discovery of all of the other members of the Src family of protein kinases. Src drug discovery has been aimed at the role of Src in oncogenesis. Indeed, most of the FDA-approved small molecule inhibitors of protein kinases are directed toward neoplastic diseases. Unlike BRAF or EGFR mutants or BCR-Abl fusion proteins, Src is not a primary driver of tumorigenesis, but rather it is a participant in many pathways promoting cell division and survival. Moreover, Src mutants in tumors are very rare. Thus, it is unlikely that anti-Src monotherapy will be efficacious in the treatment of cancers. Since Src is a participant in many aspects of cell division, invasion, migration and survival, Src inhibition may play an important auxiliary role in various cancer treatments. Now, four orally effective Src/multikinase inhibitors are FDA-approved for the treatment of various malignancies.
Understanding of Src structure and function, regulation, and localization has increased dramatically since its discovery. One-hundred years after the original description of Src, this protein continues to attract keen interest because of its multiplicity of actions in the molecular signaling pathways underlying developmental as well as oncogenic events. Many studies have addressed the molecular mechanisms of Src regulation in cells and tumor tissues. In order to clarify and fully elucidate the normal physiologic function of Src and other SFKs and to fully comprehend Src signaling networks in various cancers, Src interactions with specific targets or binding partners in different subcellular localization studies should be characterized in as much detail as possible.