scholarly journals Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases.

1996 ◽  
Vol 132 (6) ◽  
pp. 1037-1052 ◽  
Author(s):  
T Fukushima ◽  
T K Waddell ◽  
S Grinstein ◽  
G G Goss ◽  
J Orlowski ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Plasma Membranes ◽  
Kinase Inhibitors ◽  
Human Neutrophils ◽  
Cross Linking ◽  
Fcgamma Receptors ◽  
Beta2 Integrins ◽  
Cytosolic Acidification

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.

scholarly journals The role of cholesterol and sphigomyelin in tyrosine phosphorylation of proteins and capping of Fcgamma receptor II.

1999 ◽  
Vol 46 (1) ◽  
pp. 107-116 ◽  
Author(s):  
A Drzewiecka ◽  
K Kwiatkowska ◽  
A Sobota
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Fcgamma Receptor ◽  
Surface Receptors ◽  
Phosphorylation Of Proteins ◽  
Beta Cyclodextrin ◽  
Plasma Membrane Microdomains

Cross-linking of cell surface receptors by multivalent ligands, e.g. by antibodies, evokes their clustering -- patching. Subsequently, these clusters can be translocated by the acto-myosin machinery toward one pole of the cell and assembly cap. Patching of FcgammaRII in U937 cells correlates with tyrosine phosphorylation of several proteins while cap assembly correlates with their dephosphorylation. To study the mechanism of activation of tyrosine kinases during FcgammaRII activation we disturbed the organization of the putative plasma membrane microdomains by depletion of membrane cholesterol and sphingomyelin. Cholesterol was removed with the use of beta-cyclodextrin while sphingomyelin was decomposed by exogenous sphingomyelinase. Cyclodextrin at 5-10 mM removed about 70% of cholesterol from the cells and abolished the assembly of FcgammaRII caps thereby arresting the receptors at the patching stage. Similarly, 70 mU/ml sphingomyelinase inhibited cap formation by 60%. Cholesterol and sphingomyelin depletion also suppressed the tyrosine phosphorylation of proteins which accompanied cross-linking of FcgammaRII. The observations indicate that cholesterol and sphingomyelin can control the interactions of tyrosine kinases with clustered FcgammaRII.

scholarly journals Recruitment of the cross-linked opsonic receptor CD32A (FcγRIIA) to high-density detergent-resistant membrane domains in human neutrophils

2004 ◽  
Vol 381 (3) ◽  
pp. 919-928 ◽  
Author(s):  
Emmanuelle ROLLET-LABELLE ◽  
Sébastien MAROIS ◽  
Kathy BARBEAU ◽  
Stephen E. MALAWISTA ◽  
Paul H. NACCACHE
Keyword(s):  
Tyrosine Phosphorylation ◽  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
High Density ◽  
Human Neutrophils ◽  
Cross Linking ◽  
Src Kinases ◽  
Membrane Domains ◽  
Detergent Resistant Membranes

We have previously shown that CD32A (or FcγRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-β-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-β-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

scholarly journals The B cell antigen receptor of class IgD induces a stronger and more prolonged protein tyrosine phosphorylation than that of class IgM.

1995 ◽  
Vol 181 (3) ◽  
pp. 1005-1014 ◽  
Author(s):  
K M Kim ◽  
M Reth
Keyword(s):  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Antigen Receptor ◽  
Cross Linking ◽  
Protein Tyrosine Phosphorylation ◽  
Antigen Receptors ◽  
Antigen Specificity ◽  
Protein Tyrosine ◽  
Substrate Phosphorylation

Most mature B lymphocytes coexpress two classes of antigen receptor, immunoglobulin (Ig)M and IgD. The differences in the signal transduction from the two receptors are still a matter of controversy. We have analyzed B cell lines expressing IgM or IgD antigen receptors with the same antigen specificity. Cross-linking of these receptors with either antigen, or class-specific antibodies, results in the activation of protein tyrosine kinases and the phosphorylation of the same substrate proteins. The kinetic and the intensity of phosphorylation, however, was quite different between the two receptors when they were cross-linked by antigen. In membrane IgM-expressing cells, the substrate phosphorylation reached a maximum after 1 minute and diminished after 60 minutes whereas, in the membrane IgD-expressing cells, the substrate phosphorylation increased further over time, reached its maximum at 60 minutes, and persisted longer than 240 minutes after exposure to antigen. As a result, the intensity of protein tyrosine phosphorylation induced by cross-linking of membrane IgD was stronger than that induced by membrane IgM. Studies of chimeric receptors demonstrate that only the membrane-proximal C domain and/or the transmembrane part of membrane-bound IgD molecule is required for the long-lasting substrate phosphorylation. Together, these data suggest that the signal emission from the two receptors is controlled differently.

Thrombin Mediated Tyrosine Phosphorylation of PKCδ in Human Platelets Occurs in a Calcium- and Src Family Tyrosine Kinase- Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3541-3541
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Satya Kunapuli
Keyword(s):  
Tyrosine Kinase ◽  
Intracellular Calcium ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Human Platelets ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Glycoprotein Vi ◽  
Pkc Isoforms

Abstract Protein kinase C (PKC)-δ is a novel PKC that has been shown to be tyrosine phosphorylated upon stimulation with agonists in platelets. Tyrosine phosphorylation of PKCδ has been shown to occur in a Fyn-dependent manner downstream of glycoprotein VI (GPVI) signaling in platelets. Although thrombin causes tyrosine phosphorylation of PKCδ in platelets, the mechanism of this event is not elucidated. In this study, we investigated whether G-protein signaling pathways utilize similar pathways as GPVI in tyrosine phosphorylation of PKCδ. Protease activated receptor (PAR) -1 selective peptide, SFLLRN and PAR - 4 selective peptide, AYPGKF caused a time- and concentration-dependent increase in tyrosine phosphorylation of PKCδ in human platelets. However, AYPGKF failed to cause tyrosine phosphorylation of PKCδ in Gq-deficient mouse platelets. Both U73122, a phospholipase C (PLC) inhibitor, and dimethyl-BAPTA, an intracellular calcium chelator, inhibited the tyrosine phosphorylation of PKCδ downstream of the PAR activation suggesting a role for Gq/PLC pathways and intracellular calcium in mediating this event. Inhibition of PKC isoforms using GF109203X potentiated the tyrosine phosphorylation of PKCδ. The Src family tyrosine kinase inhibitors, PP1 and PP2 inhibited the tyrosine phosphorylation of PKCδ suggesting a role for Src family tyrosine kinase members in this event. We also found that both Lyn and Src are physically associated with PKCδ in a constitutive manner in platelets. Finally we found that there was a time-dependent activation of Src following activation of platelets with thrombin. Thus, the precomplexed Src and Lyn tyrosine kinases get activated following PAR stimulation resulting in the tyrosine phosphorylation of PKCδ. All these data indicate that tyrosine phosphorylation of PKCδ downstream of thrombin occurs in a calcium- and Src-family kinase dependent manner in human platelets.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3970-3970
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Class Iii ◽  
Primary Resistance

Abstract Abstract 3970 Poster Board III-906 Purpose The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. Results We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. Conclusion Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures: No relevant conflicts of interest to declare.

E3 ligase–defective Cbl mutants lead to a generalized mastocytosis and myeloproliferative disease

Blood ◽  
2009 ◽  
Vol 114 (19) ◽  
pp. 4197-4208 ◽  
Author(s):  
Srinivasa Rao Bandi ◽  
Christian Brandts ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Receptor Tyrosine Kinases ◽  
E3 Ligase ◽  
Myeloproliferative Disease ◽  
Primary Resistance ◽  
Murine Bone Marrow

Abstract Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold. We recently identified the first c-Cbl mutation in human disease in an acute myeloid leukemia patient, called Cbl-R420Q. Here we analyzed the role of Cbl mutants on Kit-mediated transformation. Coexpression of Cbl-R420Q or Cbl-70Z with Kit induced cytokine-independent proliferation, survival, and clonogenic growth. Primary murine bone marrow retrovirally transduced with c-Cbl mutants and transplanted into mice led to a generalized mastocytosis, a myeloproliferative disease, and myeloid leukemia. Overexpression of these Cbl mutants inhibited stem cell factor (SCF)–induced ubiquitination and internalization of Kit. Both Cbl mutants enhanced the basal activation of Akt and prolonged the ligand-dependent activation. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on receptor tyrosine kinases, but independent of their kinase activity. Instead, transformation depends on the Src family kinase Fyn, as c-Cbl coimmunoprecipitated with Fyn and inhibition abolished transformation. These findings may explain primary resistance to tyrosine kinase inhibitors targeted at receptor tyrosine kinases.

scholarly journals Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc epsilon RI cross-linking.

1994 ◽  
Vol 14 (8) ◽  
pp. 5108-5113 ◽  
Author(s):  
Y Kawakami ◽  
L Yao ◽  
T Miura ◽  
S Tsukada ◽  
O N Witte ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Immunoglobulin E ◽  
Cross Linking ◽  
Mouse Bone Marrow ◽  
Ionophore A23187 ◽  
Membrane Compartment ◽  
High Affinity Receptor ◽  
Protein Kinase C Activation

Tyrosine phosphorylation of several cellular proteins is one of the earliest signaling events induced by cross-linking of the high-affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells or basophils. Tyrosine kinases activated during this process include the Src family kinases, Lyn, c-Yes, and c-Src, and members of another subfamily, Syk and PTK72 (identical or highly related to Syk). Recently, some of us described two novel tyrosine kinases, Emb and Emt, whose expression was limited to subsets of hematopoietic cells, including mast cells. Emb turned out to be identical to Btk, a gene product defective in human X-linked agammaglobulinemia and in X-linked immunodeficient (xid) mice. Here we report that Fc epsilon RI cross-linking induced rapid phosphorylation on tyrosine, serine, and threonine residues and activation of Btk in mouse bone marrow-derived mast cells. A small fraction of Btk translocated from the cytosol to the membrane compartment following receptor cross-linking. Tyrosine phosphorylation of Btk was not induced by either a Ca2+ ionophore (A23187), phorbol 12-myristate 13-acetate, or a combination of the two reagents. Co-immunoprecipitation between Btk and receptor subunit beta or gamma was not detected. The data collectively suggest that Btk is not associated with Fc epsilon but that its activation takes place prior to protein kinase C activation and plays a novel role in the Fc epsilon RI signaling pathway.

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