The Tec family of tyrosine kinases transduces signals from antigen and other receptors in cells of the hematopoietic system. In particular, interleukin-2 inducible T cell kinase (Itk) plays an important role in modulating T cell development and activation. Itk is activated by receptors via a phosphatidylinositol 3-kinase-mediated pathway, which results in recruitment of Itk to the plasma membrane via its pleckstrin homology domain. We show here that membrane localization of Itk results in the formation of clusters of at least two molecules within 80 Å of each other, which is dependent on the integrity of its pleckstrin homology domain. By contrast, the proline-rich region within the Tec homology domain, SH3 or SH2 domains, or kinase activity were not required for this event. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane as only receptors that recruit phosphatidylinositol 3-kinase reside in the same membrane vicinity as the recruited Itk. Our results indicate that Itk forms dimers in the membrane and that receptors that recruit Itk do so to specific membrane regions.The Tec family of non-receptor tyrosine kinases is the second largest family of non-receptor tyrosine kinases (1). This family, which includes Itk, 2 is involved in transducing signals from a number of receptors, including the T cell receptor, B cell receptor, Fc⑀R, c-Kit, CD28, CD2, CD32, and the erythropoietin receptor (1-6). These kinases play important roles in regulating cytokine and immune receptor signals that regulate the immune response (7,8). Itk in particular is involved in early T cell receptor signaling and regulates increases in intracellular calcium and activation of the nuclear factor of activated T cells family of transcription factors (9, 10). In addition, Itk regulates the development of Th2 cells and their subsequent cytokine secretion, thereby modulating the immune response (10 -13). A common feature of these kinases is that they require the activation of PI3 kinase, as well as Src kinase activation for their activity (14 -17). These kinases, including Itk, have a PH domain that allows them to be recruited to the plasma membrane by an activated PI3 kinase (1). Thus, membrane recruitment is a critical component of their activation, and Itk can be found at the plasma membrane of cells, although other events are required for its full activity (17)(18)(19). It is, however, not clear whether this is general membrane localization of the protein in anticipation of activation or a specific localization in certain regions of the plasma membrane.The structure of Itk in cells is not known, although it has been suggested to form dimers in its inactive state, with a resultant monomerization upon activation (20,21). We have examined these issues using a split YFP system (22) and find that Itk forms dimers or higher order clusters; however, it does so only at the plasma membrane and only in the vicinity of a receptor that can recruit PI3 kinase. Our data shed new light on the regulation, struct...

Highlights d RNF5-mediated degradation of PHGDH prevents the proliferation of breast cancer cells d The acetylation of PHGDH at K58 disrupts the interaction of RNF5-PHGDH d SIRT2 and Tip60 regulate the reversible acetylation modification of PHGDH d PHGDH is negatively correlated with RNF5 in human breast cancer

Tec family tyrosine kinases transduce signals from antigen and other receptors. In particular, Itk plays an important role in T-cell development and activation. Itk has an N-terminal pleckstrin homology domain, a Tec Homology domain with a prolinerich region, SH3 and SH2 domains and a kinase domain, the structure each of which has been determined. However, the full structure of Itk and other Tec kinases remain elusive. Models of Itk suggest either a head to tail dimer, with the SH2 domain interacting with the SH3 domain, or a folded monomer with the SH3 domain interacting with the proline-rich region. We show here that in vivo Itk exists as a monomer, with the pleckstrin homology domain less than 80 Å from the C terminus. Zn 2؉ coordinating residues in the Tec Homology domain, not the proline-rich region, are critical for this intramolecular interaction. These data have implications for our understanding of Tec family kinase structure.The Tec family of non-receptor tyrosine kinases, including Itk, 2 is the second largest family of non-receptor tyrosine kinases (1). They regulate signals emanating from multiple receptors, most prominently the TcR and BcR (1-6). Itk in particular has been shown to regulate TcR signals leading to increases in intracellular calcium, ERK (extracellular signalregulated kinase)/mitogen-activated protein kinase, and activation of transcription factors NFAT and AP-1 (7, 8). More recently, it has been determined that Itk regulates the secretion of Th2 cytokines (9 -11). In addition, Itk has been shown to be involved in the development of conventional or naïve phenotype CD8 ϩ T cells, CD4 ϩ T cells and NKT cells (12-16). Itk is structurally organized into five domains, an N-terminal pleckstrin homology (PH) domain, followed by a TH domain, which contains a Zn 2ϩ -binding BH motif and one PRR, SH3 and SH2 domains, and a C-terminal kinase domain. During stimulation of the TcR, phosphatidylinositol 3-kinase is activated, resulting in the formation of cell membrane phosphoinositides, to which the PH domain of Itk binds. Itk also forms dimers specifically at the plasma membrane in the vicinity of receptors that activate phosphatidylinositol 3-kinase (17). Once Itk is recruited to the membrane, it is phosphorylated by Src family kinases (18,19). Upon activation, Itk is enriched in membrane rafts and interact with other signaling proteins through its SH2, SH3, and TH domain. Subsequently, Itk activates several downstream signaling components, including phospholipase C␥1, and regulates the Ca 2ϩ signaling pathway (9).Although the structure of each of the individual domains of Itk is known, that of the full-length protein is unknown. A number of studies have suggested that the conformation of proteintyrosine kinases is controlled by the self-interaction of domains, thus retaining them in the inactive state (20,21). Src family tyrosine kinases, which have similar overall structures to Tec kinases with the exception of the TH and PH domains, are folded via intramolecular interactions between C-termin...

Itk is a Tec family of tyrosine kinase that transduces signals from the T cell receptor and other receptors to modulate T cell development and activation. Itk is activated by receptors via a PI3 kinase mediated pathway, which results in recruitment of Itk to the plasma membrane via its PH domain. We show that membrane localization of Itk results in the formation of clusters of at least 2 molecules within 80 angstroms of each other. In addition, only the PH domain, but not the PRR, SH3 or SH2 domain, or kinase activity of Itk, is required for its formation of clusters at the plasma membrane. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane since only receptors that recruit PI3 kinase reside in the same membrane vicinity as the recruited Itk. Itk also forms dimers or clusters when associated with the adaptor protein SLP-76. Our results indicate that Itk forms membrane dimers with receptors in specific membrane regions. Furthermore, Itk forms dimers with adaptor proteins that assemble multiprotein complexes.