Adapter proteins participate in intracellular signal transduction processes through physical interaction with catalytically active binding partners resulting in the formation of signaling complexes. Incorporation of adapter proteins into these complexes is mediated by modular binding domains contained in the adapter itself or in the respective binding partner(s). Through incorporation into complexes, adapter proteins modulate the activity of catalytically active proteins, directly or indirectly. In a subtractive cDNA library screen of activated human peripheral blood CD8 T cells, a novel adapter protein was identified that was named T cell specific adapter protein (TSAd) (Spurkland et al. 1998). TSAd is also known as Src-homology 2 (SH2) domain protein 2A (SH2D2A), based upon the presence of an SH2 domain in the center of the linear sequence. Subsequently, murine TSAd was identified as an interacting partner of the Src-family protein tyrosine kinase (PTK), LCK, and the Tec family protein tyrosine kinases, RLK and ITK, in yeast two-hybrid screening experiments (Choi et al. 1999; Rajagopal et al. 1999). Hence, TSAd/SH2D2A is also known as LCK-associated adapter protein (LAD) and RLK/ITK-binding protein (RIBP).
SH2D2A resembles a classical adapter protein that contains a central SH2 domain and a carboxyl region with a conserved proline-rich stretch and tyrosine residues that are present in consensus phosphorylation motifs (Lapinski et al. 2009). The SH2 domain of SH2D2A recognizes phosphorylated tyrosine residues contained in other signaling proteins, and, visa versa, SH2 domains of other signaling proteins recognize phosphorylated tyrosine residues in the SH2D2A carboxyl region. In addition, interaction of SH2D2A with signaling proteins can be mediated by SH3 domain binding to the SH2D2A carboxyl region proline-rich stretch. The amino-terminal region of SH2D2A has no discernible structure or function.
SH2D2A is most strongly expressed in the T cell lineage and in natural killer (NK) cells (Lapinski et al. 2009). In T cells, expression levels are increased following T cell activation. SH2D2A is expressed in CD4 and CD8 T cells and comparably in CD4 T helper 1 and T helper 2 cell lines. In part, expression of SH2D2A in T cells is regulated by a cyclic adenosine monophosphate (cAMP) response element that is present in the SH2D2A promoter (Dai et al. 2004). Other than T and NK cells, SH2D2A expression has also been noted in B cells, epithelial cells, and vascular endothelial cells (Lapinski et al. 2009).
As noted above, SH2D2A interacts physically with LCK, RLK, and ITK. Furthermore, for LCK, physical interaction has been demonstrated in primary T cells between cell endogenous proteins (Marti et al. 2006). Interaction is thought to involve PTK phosphorylation of SH2D2A carboxyl region tyrosine residues that are then bound by the PTK SH2 domain. In addition, the LCK SH3 domain binds to the SH2D2A proline-rich stretch, thus constituting another mechanism of interaction.
Ligands of the SH2D2A domain that have been identified include linker of activated T cells (LAT), vascular endothelial growth factor receptor 2 (VEGFR2), and valosin-containing protein (VCP) (Lapinski et al. 2009; Marti and King 2005; Marti et al. 2001; Matsumoto et al. 2005). In the first two examples, the SH2 domain binds directly to phosphorylated tyrosine residues in the respective target proteins. In the case of VCP, however, tyrosine phosphorylation likely results in a conformational change, which exposes a distinct interaction site upon VCP for SH2D2A SH2 domain recognition.
Other signaling proteins that SH2D2A physically interacts with include Gß protein subunits of heterotrimeric G proteins and the mitogen-activated protein 3 kinase, MEKK2 (Park et al. 2007; Sun et al. 2001). However, the molecular basis for interaction with these proteins has not been clearly defined.
SH2D2A promotes activation of LCK at the outset of T cell antigen receptor (TCR) signal transduction in T cells (Marti et al. 2006). TCR recognition of ligand results in local aggregation of LCK at the plasma membrane. For those LCK molecules that preexist in an open primed conformation, aggregation leads to an increase in LCK kinase activity, consequent to LCK-mediated phosphorylation of a positive regulatory tyrosine residue present in the LCK kinase domain (Palacios and Weiss 2004). Increased LCK kinase activity then results in SH2D2A tyrosine phosphorylation and subsequent interaction of SH2D2A with the LCK SH2 and SH3 domains. These interactions are important since LCK SH2 domain binding to a phosphorylated negative-regulatory tyrosine residue, positioned carboxyl to the LCK kinase domain, and LCK SH3 domain binding to an LCK proline stretch, present in a linker region between the SH3 domain and kinase domain, act to constrain this PTK in an inactive conformation (Palacios and Weiss 2004). Therefore, in binding to LCK, SH2D2A disrupts these intramolecular inhibitory interactions. Consequently, additional LCK molecules are driven into open primed conformations whereupon they may become fully activated by phosphorylation upon the kinase domain positive-regulatory tyrosine residue. In turn, this will result in further SH2D2A phosphorylation, leading to further LCK activation and so on.
The importance of SH2D2A in the activation of LCK during the course of TCR signal transduction is shown by the finding that TCR-induced activation of LCK is impaired in T cells from SH2D2A-deficient mice (Marti et al. 2006). Consequently, all downstream signaling events in T cells including activation of the ZAP-70 PTK, activation of the Ras-MAPK pathway, calcium mobilization, and cytokine synthesis are reduced in magnitude in SH2D2A-deficient T cells.
T cell migration induced by the chemokines CXC12 and CCL5 is also SH2D2A-dependent (Park et al. 2007). Interestingly, the function of SH2D2A in this response again appears to relate to a required role for SH2D2A in the activation of LCK and ZAP-70 in the chemokine-induced signaling pathway downstream of heterotrimeric G proteins.
SH2D2A may also have a nuclear role in T cells (Marti and King 2005; Marti et al. 2001). This conclusion is based upon the finding that SH2D2A is actively transported to the nucleus in T cells via a mechanism that involves SH2 domain recognition of VCP. The exact function of SH2D2A in the cell nucleus is unknown.
Outside of the T cell compartment, expression of SH2D2A has been shown to be required for VEGFR2-induced migration of blood vascular endothelial cells in vitro and for blood vessel angiogenesis toward tumors in vivo (Matsumoto et al. 2005). Physical interaction of SH2D2A with the VEGFR2 cytoplasmic domain is necessary for both of these responses. In epithelial cell lines, SH2D2A physical interaction with MEKK2 is required for activation of the ERK5 big MAPK induced by the epidermal growth factor (EGFR), osmotic and oxidative stress (Sun et al. 2001, 2003).
SH2D2A-Deficient Mouse Model
SH2D2A-deficient mice are viable and initially healthy (Rajagopal et al. 1999). T cell development proceeds normally, and in young adult animals, numbers and ratios of peripheral T cell subsets are similar to those observed in control mice. However, with increasing age, SH2D2A-deficient mice show signs of systemic lupus-like autoimmunity (Drappa et al. 2003; Marti et al. 2005). Disease is associated with splenomegaly, increased numbers of activated memory phenotype T cells in spleen and lymph nodes, production of autoantibodies against DNA and other nuclear targets, glomerulonephritis, and leukocytic infiltration into nonlymphoid organs. Furthermore, young SH2D2A-deficient mice are highly susceptible to experimentally induced lupus-like autoimmune disease. The likely cause of autoimmunity in SH2D2A-deficient mice is impaired apoptotic death of autoreactive peripheral T cells upon recognition of self antigens (Drappa et al. 2003). The molecular basis for this impaired death is uncertain but likely relates to impaired cytokine production by SH2D2A-deficient T cells.
SH2D2A is an SH2 domain-containing signaling adapter protein that is strongly expressed in T lymphocytes. SH2D2A functions at the beginning of the TCR signal transduction cascade by promoting activation of the LCK Src-family PTK through competitive inhibition of LCK intramolecular inhibitory interactions. In addition, roles for SH2D2A in T cell migration, VEGFR2 signal transduction in endothelial cells, and activation of the big MAPK signaling pathway in epithelial cells have been described. SH2D2A-deficient mice are susceptible to the development of systemic lupus-like autoimmune disease, thus illustrating the importance of SH2D2A as a regulator of T cell tolerance to self antigens.
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