Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P 3 ) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cγ (PLCγ) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P 3 regulates receptordependent calcium signals through the function of Tec kinases.
Bruton's tyrosine kinase (Btk) is essential for B‐lineage development and represents an emerging family of non‐receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B‐cell antigen receptor (BCR) cross‐linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk‐deficient human B‐cell lines. Btk and phospholipase Cγ (PLCγ) co‐expression resulted in tyrosine phosphorylation of PLCγ and required the same Btk domains as those for Btk‐dependent calcium influx. Receptor‐dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)‐sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg‐sensitive, IP3‐gated calcium store(s) that regulates store‐operated calcium entry. Overexpression of dominant‐negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co‐cross‐linking of the BCR and the inhibitory receptor, FcγRIIb1, completely abrogated Btk‐dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.
Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotypeselective HDAC inhibitor. We have developed an isotypeselective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted. [Mol Cancer Ther 2008;7(4):759 -68]
Bruton's tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-␥. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-␥ and Src family kinases.Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase that contains a pleckstrin homology (PH) domain but no apparent lipid modification motif (1). Btk is critical for development and signaling. Btk mutations are associated with the genetic diseases human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid; refs. 2-5). XLA patients have a dramatic decrease in the number of mature B cells and circulating Ig levels (6). Xid mice or mice with a targeted disruption of Btk have diminished B cell numbers and levels of certain Ig classes (7-9).PH domains are primarily involved in protein-protein or protein-lipid interactions and regulate enzyme function by controlling interacting partners or cellular localization (10,11). The N-terminal PH domain of Btk is essential for its activation and biological activity. A mutation in the Btk PH domain causes Xid (R28C; refs. 4 and 5), and other mutations within the PH domain also result in XLA (12, 13). In contrast, a Glu-to-Lys mutation (E41K, BTK*) in the PH domain activates Btk and increases membrane association (14). These gain or loss of function mutations suggest that the PH domain is a critical regulatory domain for Btk activation but give little information regarding specific signaling mechanisms.The PH domain of Btk was recently shown to bind the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (15, 16) and inositol 3-phosphates in vitro (17). Computer modeling identified several residues within the Btk PH domain including Lys-12, Phe-25, and Arg-28, which are thought to be essential for binding these lipid molecules (15,16,18,19). Interestingly, mutation of these residues results in human XLA (e.g., F25S and R28H; ref. 12) or murine Xid (R28C...
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