Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in <i>Escherichia coli</i>

Wang, Cheng C.; Moriyama, Ryuichi; Low, Philip S.; Badylak, J; Dixon, Jack E.; Lux, Samuel E.

doi:10.1002/pro.5560010913

Cited by 48 publications

(37 citation statements)

References 35 publications

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“…All 3 antibodies gave similar results. Similar results were also obtained when cdb3, another substrate with Syk specificity, was used in this assay 37 (data not shown).…”

Section: Org Fromsupporting

confidence: 86%

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Rapid ubiquitination of Syk following GPVI activation in platelets

Dangelmaier

Quinter

Jin

et al. 2005

Blood

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Spleen tyrosine kinase (Syk) activation is a key intermediate step in the activation of platelets by the physiologic agonist collagen. We have found that Syk is rapidly ubiquitinated upon activation of platelets by collagen, collagen-related peptide (CRP), and convulxin. The Src family kinase inhibitors prevented Syk phosphorylation and its ubiquitination, indicating that the process is downstream of Src kinases. The ubiquitination of Syk did not cause degradation of the protein as evidenced by the lack of effect of proteasomal and lysosomal inhibitors. We separated ubiquitinated Syk from its nonubiquitinated counterpart and used an in vitro kinase assay to compare their activities. We found that the ubiquitinated Syk appeared to be about 5-fold more active. Using a phosphospecific antibody to Syk (Tyr525/Tyr526) that measures activated Syk, we found that most (60%-75%) of the active Syk is in the ubiquitinated fraction. This result explains the apparent high specific activity of ubiquitinated Syk. In c-Cbl-deficient mice, Syk is not ubiquitinated, implicating c-Cbl as the E3 ligase involved in Syk ubiquitination. Furthermore, Syk is not dephosphorylated in these mice. We propose that c-Cbl plays a regulatory role in glycoprotein VI (GPVI)/Fc receptor ␥ (FcR␥)-chain-dependent platelet activation through its interaction with Syk. IntroductionThe activation of platelets by collagen is an essential event in prevention of bleeding. 1 Collagen binds to at least 2 different receptors on the platelet membrane. One receptor is an integrin ␣2␤1 that is primarily responsible for firm adhesion of platelets to collagen. 2 The other is the glycoprotein VI (GPVI)/Fc receptor ␥ (FcR␥)-chain complex that is responsible for the majority of the intracellular signaling events. 3,4 Platelet activation by collagen leads to an increase in intracellular Ca 2ϩ and activation of protein kinase C. 5 The responses are dependent on the activation of phospholipase C␥2 (PLC␥2) downstream of GPVI/FcR␥-chain activation. [6][7][8] While the details of signaling through this receptor are not complete, many aspects have been described and the pathways parallel activation by immune receptors in lymphocytes. [9][10][11] Initially the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR␥-chain becomes phosphorylated by an Src family kinase, Fyn and/or Lyn. The spleen tyrosine kinase (Syk) binds to the ITAM and becomes autophosphorylated. 12 Activation of Syk leads to phosphorylation of several adapter proteins such as linker for T-cell activation (LAT) and Src homology 2-containing leukocyte protein 76 (SLP76), activation of phosphatidylinositol 3-kinase (PI-3 kinase), recruitment of Bruton tyrosine kinase (Btk), and ultimately activation of PLC␥2 by tyrosine phosphorylation. 13,14 Activation of PLC␥2 in vitro has been shown to depend on phosphorylation on at least 2 tyrosines. 8 c-Cbl is a multidomain adapter protein that is highly expressed in hematopoietic cells and has been shown to facilitate signal transduction in several signalin...

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“…All 3 antibodies gave similar results. Similar results were also obtained when cdb3, another substrate with Syk specificity, was used in this assay 37 (data not shown).…”

Section: Org Fromsupporting

confidence: 86%

“…Cdb3 was expressed in Escherichia coli and purified on a His-select HC-nickel column (Sigma). 37 SuperSignal West Pico chemiluminescent substrate was from Pierce (Rockford, IL). Convulxin was purified according to the method of Polgar et al 31 Collagen (type I, equine tendon) was from Chronolog (Havertown, PA).…”

Section: Methodsmentioning

confidence: 99%

Rapid ubiquitination of Syk following GPVI activation in platelets

Dangelmaier

Quinter

Jin

et al. 2005

Blood

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“…This construct was used to transform Escherichia coli BL21 (DE3)(pLysS) (Novagen), and expression and purification of cfb3 were performed essentially as described (34). Purified bovine brain tubulin (containing a mixture of ␣ and ␤ tubulin) was a gift of Dr. E. Hamel (NCI, Bethesda, MD).…”

Section: Methodsmentioning

confidence: 99%

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

Isakov

Wange

Watts

et al. 1996

Journal of Biological Chemistry

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The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn 2؉ over Mg 2؉ . The apparent K m of ZAP-70 for ATP was ϳ3.0 M. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Several proteins were tested as potential in vitro substrates of ZAP-70. Only ␣-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting K m values of ϳ3.3 and ϳ2.5 M, respectively ([ATP] ‫؍‬ 50 M). ␣-and ␤-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCR chain or short peptides corresponding to the CD3⑀ or the TCR immunoreceptor tyrosinebased activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.Stimulation of the antigen-specific receptor on T cells (TCR) 1 initiates a cascade of biochemical events leading to activation of the mature T cell. Among the earliest biochemical events that follows TCR triggering is the activation of protein-tyrosine kinases (PTKs) resulting in a transient tyrosine phosphorylation of multiple intracellular protein substrates (reviewed in Refs. 1 and 2) including the TCR/CD3 subunits (3-5). These multiple TCR subunits lack intrinsic PTK activity; instead, they interact with and activate cytoplasmic PTKs that couple the receptor to the signaling apparatus.PTKs that have been implicated in TCR signaling include the Src family members, Fyn and Lck, and the TCR -associated-protein (ZAP-70) (reviewed in Refs. 1 and 2). Although Fyn is constitutively associated with nonpolymorphic components of TCR/CD3 subunits (6, 7), Lck is noncovalently associated with the cytoplasmic domain of the CD4 and CD8 co-receptor molecules (8, 9). Both Fyn and Lck can also directly associate with the inner leaflet of the plasma membrane via their Nterminal myristylation/palmitylation signals (10), whereas ZAP-70, which lacks this sequence, is thought to reside in the cytosol in resting cells and then becomes rapidly recruited to the TCR upon its activation and phosphorylation (11,12). Following interaction of the TCR with peptide-bound MHC molecules on the surface of antigen presenting cells, CD4 or CD8 interact ...

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“…The plasmid for the expression of the human cytoplasmic domain of erythrocyte band 3 protein (cdb3) was provided by Dr. P. S. Law (Purdue University, West Lafayette, IN). The preparation of cdb3 was previously described (30,37). Biotinylated diphosphorylated peptide based on the ITAM of Fc⑀RI␥ (biotin-␥PP) was previously described (38).…”

Section: Materials and Absmentioning

confidence: 99%

Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function

Sada

Zhang

Siraganian

2000

The Journal of Immunology

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The protein tyrosine kinase Syk plays an essential role in FcεRI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the FcεRI-mediated tyrosine phosphorylation of phospholipase C-γ and a dramatic enhancement of histamine release. The in vivo FcεRI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the FcεRI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The FcεRI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.

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Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli

Cited by 48 publications

References 35 publications

Rapid ubiquitination of Syk following GPVI activation in platelets

Rapid ubiquitination of Syk following GPVI activation in platelets

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

Point Mutation of a Tyrosine in the Linker Region of Syk Results in a Gain of Function

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