Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

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.

NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcεRI). In this study, we have made the following observations on growth properties and FcεRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcεRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-γ1 and C-γ2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcεRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-α and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Cα and protein kinase CβII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.

Akt ‫؍(‬ protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKC␤II, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKC␤ is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKC␤II appears to work as a cell type-and stimulusspecific PDK2.Akt is a subfamily of the AGC serine/threonine kinases consisting of Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥. These kinases have an N-terminal pleckstrin homology domain followed by a C-terminal kinase catalytic domain. Numerous studies implicate Akt in survival, proliferation, and glucose metabolism (reviewed in Refs. 1-3). Akt activation requires the recruitment of the enzyme to the plasma membrane through the interaction of the pleckstrin homology domain with plasma membrane-bound lipid products of phosphatidylinositol 3-kinase, i.e. phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (4 -7). Membrane-bound Akt is then phosphorylated at two sites for full activation (8); Thr-308 in the activation loop of the kinase domain is phosphorylated by PDK1 1 (5, 7, 9) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been controversial (10), although several PDK2 candidates including integrin-linked kinase (ILK) (11, 12) and autophosphorylation (13) have been proposed. PKC is also a subfamily of the AGC serine/threonine kinases, which are involved in proliferation, differentiation, metabolism, and cell-type specific functions (14, 15). Based on structural features and cofactor requirements, PKC isoforms are classified into three categories; conventional or classical isoforms (cPKCs: ␣, ␤I, ␤II, and ␥) depend on diacylglycerol (DAG) and Ca 2ϩ for their activation, novel isoforms (nPKCs: ␦, ⑀, , and ) depend on DAG, but not Ca 2ϩ , and atypical isoforms (atypical PKC isoforms: and /) do not require either DAG or Ca 2ϩ .Cross-linking of high-affinity IgE receptors (Fc⑀RI) expressed on the surface of mast cells activates Akt (16) and various PKC isoforms including PKC␤I and PKC␤II (17). Importantly, Akt in mast cells is involved in Fc⑀RI-induced production of cytokines such as IL-2 through the activation of such trans...

Pre-B cell receptor (pre-BCR) expression is critical for B lineage development. The signaling events initiated by the pre-BCR, however, remain poorly defined. We demonstrate that lipid rafts are the major functional compartment for human pre-B cell activation. A fraction of pre-BCR was constitutively raft associated, and receptor engagement enhanced this association. These events promoted Lyn activation and Igbeta phosphorylation and led to the generation of a raft-associated signaling module composed of tyrosine phosphorylated Lyn, Syk, BLNK, PI3K, Btk, VAV, and PLCgamma2. Formation of this module was essential for pre-BCR calcium signaling. Together, these observations directly link the previously identified genetic requirement for the components of this module in B lineage development with theirfunctional role(s) in human preBCR signaling.

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.slow growth | infectious disease | metabolic heterogeneity | cystic fibrosis | hydrogen isotope labeling

Purpose We sought a human blood T2-oximetery calibration curve over the wide range of hematocrits commonly found in anemic patients applicable with T2 Relaxation Under Spin Tagging (TRUST). Methods Blood was drawn from 5 healthy control subjects. Ninety-three in vitro blood transverse relaxation (T2b) measurements were performed at 37° C over a broad range of hematocrits (10–55%) and oxygen saturations (14–100%) at 3T. In vivo TRUST was performed on 35 healthy African American control subjects and 11 patients with chronic anemia syndromes. Results 1/T2 rose linearly with hematocrit (r2 = 0.96), for fully saturated blood. Upon desaturation, 1/T2 rose linearly with the square of the oxygen extraction, (1−Y)2, and the slope was linearly proportional to hematocrit (r2= 0.88). The resulting bilinear model between 1/T2, (1−Y)2 and hematocrit had a combined r2 of 0.96 and a coefficient of variation of 6.1%. Using the in-vivo data, the bilinear model had significantly lower bias and variability than existing calibrations, particularly for low hematocrits. In-vivo Bland Altman analysis demonstrated clinically relevant bias that was −6% (absolute saturation) for hematocrits near 30% and rose to +6% for hematocrits near 45%. Conclusion This work introduces a robust bilinear calibration model that should be used for MRI oximetry.