Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced by in vitro incubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Syk in vitro and in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.

Role of Syk in B-cell development and antigen-receptor signaling

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles—promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79α/β BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

Adult brain retains the potential to generate oligodendroglial progenitors with extensive myelination capacity

Remyelination of focal areas of the central nervous system (CNS) in animals can be achieved by transplantation of glial cells, yet the source of these cells in humans to similarly treat myelin disorders is limited at present to fetal tissue. Multipotent precursor cells are present in the CNS of adult as well as embryonic and neonatal animals and can differentiate into lineage-restricted progenitors such as oligodendroglial progenitors (OPs). The OPs present in adults have a different phenotype from those seen in earlier life, and their potential role in CNS repair remains unknown. To gain insights into the potential to manipulate the myelinating capacity of these precursor and/or progenitor cells, we generated a homogenous culture of OPs from neural precursor cells isolated from adult rat subependymal tissues. Phenotypic characterization indicated that these OPs resembled neonatal rather than adult OPs and produced robust myelin after transplantation. The ability to generate such cells from the adult brain therefore opens an avenue to explore the potential of these cells for repairing myelin disorders in adulthood.

Dynamic regulation of c-Jun N-terminal kinase activity in mouse brain by environmental stimuli

Activation of the recently identified c-Jun N-terminal kinases (JNKs) typically results in programmed cell death (apoptosis) in neurons and other cell types grown in culture. However, the effects of JNK activation in the central nervous system in vivo are unknown. At baseline, JNK activity in mice was on average 17-fold higher in brain than in peripheral organs, whereas JNK protein levels were similar. In brain, JNK was expressed primarily in neurons. Restraining mice or allowing them to explore a novel environment rapidly increased JNK activity 3- to 15-fold in various brain regions, but these manipulations did not increase brain activity of the extracellular signal-regulated kinase. Because noninvasive environmental stimuli that do not induce neurodegeneration elicited prominent increases in JNK activity in the brain, we conclude that acute activation of the JNK cascade in central nervous system neurons does not induce neuronal apoptosis in vivo. In contrast, the high baseline activity of JNK in the brain and the activation of the JNK cascade by environmental stimuli suggest that this kinase may play an important physiological role in neuronal function.

Characterization of a calmodulin kinase II inhibitor protein in brain

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII α and β and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i.e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.

Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain

Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35−/− mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35−/−Cdk5 +/− mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35−/− mice compared with Cdk5−/− mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35−/− causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.

Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.

MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia

The MEK1 (MAP kinase/ERK kinase)/ERK (extracellular-signal-responsive kinase) pathway has been implicated in cell growth and differentiation [Seger, R. & Krebs, E. G. (1995) FASEB J. 9, 726–735]. Here we show that the MEK/ERK pathway is activated during focal cerebral ischemia and may play a role in inducing damage. Treatment of mice 30 min before ischemia with the MEK1-specific inhibitor PD98059 [Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. & Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489–27494] reduces focal infarct volume at 22 hr after ischemia by 55% after transient occlusion of the middle cerebral artery. This is accompanied by a reduction in phospho-ERK1/2 immunohistochemical staining. MEK1 inhibition also results in reduced brain damage 72 hr after ischemia, with focal infarct volume reduced by 36%. This study indicates that the MEK1/ERK pathway contributes to brain injury during focal cerebral ischemia and that PD98059, a MEK1-specific antagonist, is a potent neuroprotective agent.

A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5

CD38 ligation on mouse B cells by CS/2, an anti-mouse CD38 mAb, induced proliferation, interleukin 5 (IL-5) receptor α chain expression, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) from wild-type, but not from X chromosome-linked, immunodeficient mice. B cells from fyn-deficient (Fyn−/−) and lyn-deficient (Lyn−/−) mice showed an impaired response to mAb CS/2 for proliferation and IL-5 receptor α chain expression, and B cells from fyn/lyn double-deficient (Fyn/Lyn−/−) mice did not respond at all to mAb CS/2. The Btk activation by CD38 ligation was observed in B cells from Fyn−/− mice, and it was severely impaired in B cells from Lyn−/− and Fyn/Lyn−/− mice. CD38 expression on B cells from three mutant strains was comparable to that on control B cells. We infer from these results that both Fyn and Lyn are required and that their signals are synergistic for B cell triggering after CD38 ligation. Lyn is upstream of Btk activation in the CD38 signaling. Stimulation of B cells with IL-5 together with CD38 ligation induces not only IgM but also IgG1 secretion. Analysis of the synergistic effects of IL-5 and CD38 ligation on IgG1 secretion revealed the impaired IgG1 secretion of B cells from Lyn−/− and Fyn/Lyn−/− mice. These data imply that Lyn is involved in B cell triggering by CD38 ligation plus IL-5 for isotype switching.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

Phosphorylation of two regulatory tyrosine residues in the activation of Bruton’s tyrosine kinase via alternative receptors

Mutation of Bruton’s tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fcɛ receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosine-phosphorylated. Phosphorylated Btk comprised only a small fraction (≤5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.