SH2 domains

Tyrosine Phosphorylation and SH2 domains

Tyrosine phosphorylation is an essential event in transforming many extracellular signals (e.g. from cytokines, chemokines, growth factors) into intracellular signals, leading for example to changes in gene expression. Phosphorylation of tyrosine residues is a relatively rare event (0.01 – 1.0 % of all protein phosphorylation events), but it is of great interest because its deregulation has been firmly established for several human diseases, including many cancers.

After binding of a suitable ligand to the extracellular domain of a transmembrane receptor, the intrinsic or associated tyrosine kinase becomes activated allowing signalling proteins, such as adapter proteins and various signalling enzymes to bind and become regulated, usually resulting in the generation of several intracellular signals.

The SH2 (Src homology 2) domain is a structurally conserved protein domain which evolved early during metazoan evolution and is critical for the recognition of numerous phosphorylated tyrosine residues (pTyr). It is present in many proteins involved in intracellular signal transduction pathways, such as tyrosine kinases and adapter proteins, and allows them to bind to specific peptide motifs containing a phosphorylated tyrosine residue. SH2 domains are usually 100-120 amino acids in length and fold into a stable modular structure composed of two alpha-helices that sandwich an anti-parallel beta-sheet consisting of seven beta-strands. 120 SH2 domains have been detected in 115 human proteins so far. They hence represent the largest class of pTyr-recognition domains.

Applications

Detection of SH2 binding pY-proteins in cell lysates after precipitation (pulldowns)

Mass spectrometric identification of SH2 binding pY-proteins

SH2 Far Western Blot with total cell lysates

SH2 Far Western Blot after SH2-mediated precipitation of pY-proteins from cell lysates

SH2 domain histochemistry (HC) with cell pellets (cytospins), fresh frozen tissue, or formalin-fixed paraffin embedded (FFPE) tissue blocks


	pY-Hunter-SH2-Kit sheet


				*Our pY-Hunter SH2 domain kits contain the following components:* 1. GST negativ control 2. GST-SH2 domain 3. GST-capture beads 4. OPTI-Ex-pY cell extraction buffer 5. SDS-PAGE sample buffer 6. FREE: 400 µl of high performance anti-pTyr mAb (introductory offer – as long as stocks last) *How many samples are processable with one kit containing 200 µg of SH2-GST-fusion protein?* Depending on your kind of samples a kit is typically suitable for approximately 5 to 10 pulldown reactions (with approx. 500 µg protein extract each). Alternatively, ca. 2 to 4 pulldowns with subsequent mass spectrometric analysis are possible. Far Western-Blots can be done with a wide range of probe concentrations. We suggest to test initially 5 µg/ml and to use our special pY-Hunter SH2 Far Western-Blot Block buffer [Inquire for details info@MabTag.com]




	We offer 16 different kits with recombinant SH2 domains expressed as GST-fusion proteins of the following human proteins: Designation according to current HGNC nomenclature (http://www.genenames.org/). Frequently used other names are also indicated. Where a protein contains more than one SH2 domain the relevant SH2 is specified. ABL1 / c-Abl ABL1 is a ubiquitously expressed non-receptor tyrosine kinase which is located in the cytoplasm and nucleus. It is involved in cell differentiation, cell division, cell adhesion, and stress response. ABL1 interacts via its SH2 domain with various cellular targets including PDGFR and integrins. Its activity is negatively regulated by its SH3 domain and deletion of its SH3 domain turns ABL1 into an oncogene. The t(9;22) chromosome translocation results in a fusion of BCR and ABL1 and is a hall mark of various leukemias. BLK (B lymphoid tyrosine kinase) BLK is a member of the SRC family of protein tyrosine kinases and is involved in B-cell receptor signalling. BLK also stimulates insulin synthesis and secretion in response to glucose and enhances the expression of several pancreatic beta-cell transcription factors. BTK (Bruton’s tyrosine kinase) BTK plays a crucial role in B cell development. Mutations in this gene cause X-linked agammaglobulinemia type 1, which is an immunodeficiency characterized by the failure to produce mature B lymphocytes, and which is associated with a failure of Ig heavy chain rearrangement. BLNK (B cell linker) / SLP-65 BLNK is an adaptor protein that plays a crucial role in B cell development. It links B cell receptor-associated kinase activation with downstream signalling events, thereby affecting various B cell functions. Mutations in this gene cause hypoglobulinemia and loss of B cells as well as a disease in which the pro- to pre-B-cell transition is developmentally blocked. Deficiency in this protein has also been shown in some cases of pre-B acute lymphoblastic leukemia. CRKL (Crk-like, v-crk sarcoma virus CT10 oncogene homolog (avian)-like) CRKL is a member of the CRK adapter protein family and consists of one SH2 and two SH3 domains. CRKL is expressed in many cell types, including lymphocytes and involved in negative regulation of signal transduction. It is also the major substrate of the Bcr-Abl oncokinase in CML. FGR (Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog) FGR is a member of the SRC family of protein tyrosine kinases and primarily expressed by hematopoietic cell lineages including NK cells, B cells, and differentiated myeloid cells. FGR is a critical mediator of cell membrane signalling from receptors lacking intrinsic tyrosine kinases activity, such as integrin, BCR, and Fc receptors. It functions as a negative regulator of cell migration and adhesion and infection with Epstein-Barr virus results in its overexpression. FYN (SYN, FLK, FYN oncogene related to SRC, FGR, YES) FYN is a member of the SRC family of protein tyrosine kinases. It has been implicated in T-cell receptor signalling, cell adhesion mediated signalling, and cell growth. In addition, FYN has been implicated in positive regulation of mast cell degranulation and cytokine production. FYN associates, for example, with the p85 subunit of phosphatidylinositol 3-kinase (PIK3RI) and interacts with the FYN-binding protein. GRB2 (growth factor receptor binding protein 2) GRB2 is involved in many receptor signalling pathways and consists of one SH2 domain which is flanked by two SH3 domains. In response to site-specific tyrosine phosphorylation, GRB2 is recruited to various intracellular proteins such as the EGFR cytoplasmic domain, the large docking proteins GAB1, GAB2 and IRS1, and the SHC1 adapter protein via its SH2 domain. It is also important for coupling antigen receptors to downstream signals. HCK (hemopoietic cell kinase) HCK is a member of the SRC family of protein tyrosine kinases. It is primarily expressed in cells of the myeloid and B-lymphoid lineages and has been reported to be involved in cell differentiation and proliferation as well as in neutrophil migration and degranulation. Deregulation of HCK has been associated with acute lymphocytic leukemia. LCK (lymphocyte-specific protein tyrosine kinase) Lck is a member of the SRC family of protein tyrosine kinases. It plays a crucial role in T-cell activation and differentiation by binding to cell surface receptors, including CD4 and CD8. Mutations in the LCK gene have been associated with T-cell acute lymphoblastic leukemia. LYN (v-yes-1 Yamaguchi sarcoma viral related oncogene homolog) LYN is a member of the SRC family of protein tyrosine kinases. It is expressed in hematopoietic cell lineages and is involved in cell migration and B cell development as well as in negative regulation of mast cell degranulation and cytokine production. In patients suffering from CML, LYN kinase activity is resistant to the chemotherapeutic drug imatinib. NCK1 (Nck, Nck alpha, Nck adapter protein 1) NCK1 is ubiquitously expressed and consists of three SH3 domains and one C-terminal SH2 domain. It binds, for example, to the PDGF receptor and other tyrosine-phosphorylated proteins via its SH2 domain and is involved in the regulation of cytoskeletal dynamics. PIK3R1 (phosphoinositide-3-kinase, regulatory subunit 1) / p85alpha PIK3R1 is the regulatory subunit of 85 kDa of phosphatidylinositol 3-kinase (PI3K). PI3K is a critical mediator of receptor tyrosine kinase signalling. In response to extracellular signals (e.g. growth factors, hormones) PIK3R1 is recruited via two internal SH2 domains to site-specific phosphotyrosine residues on the cytoplasmic tails of multiple receptor tyrosine kinases including EGFR, PDGFR, and IGF/IRS. Downstream signalling through the PI3K-Akt signalling cascade elicits numerous cellular events such as cell survival, cell cycle regulation, cell growth, translation and cell migration. Deregulation of PI3K has been associated in multiple human cancers and metabolic disorders. p85 contains two SH2 domains with similar phosphopeptide binding specificity. The N-terminal SH2 domain (SH2N) kit is currently available. RASA1 (RAS p21 protein activator (GTPase activating protein) 1), p120RasGap, GAP RASA1 is a member of the GAP1 family of GTPase-activating proteins and located in the cytoplasm. It stimulates the GTPase activity of normal RAS p21 but not its oncogenic counterpart. Modulation of RAS p21 activity is instrumental in regulating multiple cellular events, including cell proliferation and differentiation. In response to extracellular signals RASA1 is recruited via two internal SH2 domains to phosphotyrosine-containing proteins such as PDGFR and EGFR. Mutations leading to changes in the binding sites of this protein are associated with basal cell carcinomas. Alternative splicing results in two isoforms where the shorter isoform, lacking the N-terminal hydrophobic region but retaining the same activity, appears to be abundantly expressed in placental but not adult tissues. The N-terminal SH2 domain (SH2N) kit is currently available. SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), c-Src SRC was the first protein tyrosine kinase to be identified and is highly similar to the v-src gene of Rous sarcoma virus. It is a widely expressed proto-oncogene and is involved in cell growth and differentiation as well as in embryonic development. The deregulation of Src has been associated with advanced metastatic colon cancer and other cancer types. YES1 (v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1), c-Yes YES is a widely expressed member of the Src family of protein tyrosine kinases and a homolog of the Yamaguchi sarcoma virus oncogene. YES1 has been shown to interact with several signalling proteins, for example JAK2, CTNND1, RPL10 and Occludin.