Marietta L. Harrison

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Molecular Basis for a Direct Interaction between the Syk Protein-tyrosine Kinase and Phosphoinositide 3-Kinase

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcγRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.

Visualization of Syk-Antigen Receptor Interactions Using Green Fluorescent Protein: Differential Roles for Syk and Lyn in the Regulation of Receptor Capping and Internalization

The cross-linking of the B cell Ag receptor (BCR) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated, protein tyrosine kinases of both the Src and Syk families. To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking, we expressed in Syk-deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein. Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane. The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell. Syk is not demonstrably associated with the internalized receptor. Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane. Lyn is not required for the recruitment of Syk to the cross-linked receptor, but is required for the internalization of the clustered BCR complexes. In the absence of Lyn, receptor-Syk complexes at the plasma membrane are long lived, and the receptor-mediated activation of the NF-AT transcription factor is enhanced. Thus, Lyn appears to function to negatively regulate aspects of BCR-dependent signaling by stimulating receptor internalization and down-regulation.

The Annonaceous acetogenin bullatacin is cytotoxic against multidrug-resistant human mammary adenocarcinoma cells

Cytotoxic effects of the Annonaceous acetogenin, bullatacin, were studied in multidrug-resistant (MDR) human mammary adenocarcinoma (MCF-7/Adr) cells vs. the parental non-resistant wild type (MCF-7/wt) cells. Bullatacin was effectively cytotoxic to the MCF-7/Adr cells while it was more cytostatic to the MCF-7/wt cells. ATP depletion is the mode of action of the Annonaceous acetogenins, and these agents offer a special advantage in the chemotherapeutic treatment of MDR tumors that have ATP-dependent mechanisms.

Regulation of signaling in B cells through the phosphorylation of Syk on linker region tyrosines: A mechanism for negative signaling by the Lyn tyrosine kinase

The B cell antigen receptor (BCR) is coupled to the mobilization of Ca2+ by the protein-tyrosine kinase, Syk. Syk, recruited to the clustered BCR, becomes phosphorylated on three tyrosines (Tyr-317, Tyr-342, and Tyr-346) located within the linker region that separates the C-terminal catalytic domain from the N-terminal tandem Src homology 2 domains. Phosphorylation within the linker region can be either activating or inhibitory to Ca2+ mobilization depending on the sites that are modified. Syk that is not phosphorylated on linker region tyrosines couples the BCR to Ca2+ mobilization through a phosphoinositide 3-kinase-dependent pathway. The phosphorylation of Tyr-342 and -346 enhances the phosphorylation and activation of phospholipase C-γ and the early phase of Ca2+ mobilization via a phosphoinositide 3-kinase-independent pathway. The phosphorylation of Tyr-317 strongly dampens the Ca2+ signal. In cells that lack the Src family kinase, Lyn, the phosphorylation of the inhibitory Tyr-317 is suppressed leading to elevated production of inositol 1,4,5-trisphosphate and an amplified Ca2+ signal. This provides a novel mechanism by which Lyn functions as an inhibitor of BCR-stimulated signaling. Thus, Syk and Lyn combine to determine the pathway through which the BCR is coupled to Ca2+mobilization as well as the magnitude and duration of the Ca2+ flux.

Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.

Nucleocytoplasmic Trafficking of the Syk Protein Tyrosine Kinase

ABSTRACT The protein tyrosine kinase Syk couples the B-cell receptor (BCR) for antigen to multiple intracellular signaling pathways and also modulates cellular responses to inducers of oxidative stress in a receptor-independent fashion. In B cells, Syk is found in both the nuclear and cytoplasmic compartments but contains no recognizable nuclear localization or export signals. Through the analysis of a series of deletion mutants, we identified the presence of an unconventional shuttling sequence near the junction of the catalytic domain and the linker B region that accounts for Syks subcellular localization. This localization is altered following prolonged engagement of the BCR, which causes Syk to be excluded from the nucleus. Nuclear exclusion requires the receptor-mediated activation of protein kinase C and new protein synthesis. Both of these processes also potentiate the activation of caspase 3 in cells in response to oxidative stress in a manner that is dependent on the localization of Syk outside of the nucleus. In contrast, restriction of Syk to the nucleus greatly diminishes the stress-induced activation of caspase 3.

Renaturation and assay of protein kinases after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels

Publisher Summary This chapter presents a simple and convenient method for the in situ (in the gel) renaturation and assay of protein kinases. Proteins are separated by electrophoresis through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), renatured by removal of the SDS, and incubated with buffer containing [ γ - 32 P] adenosine triphosphate (ATP). The phosphorylation of substrates polymerized into the gel or the autophosphorylation of individual kinases is detected by autoradiography. Individual protein bands may be excised from the gel for quantification of radioactivity, analysis of phosphoamino acid content, or use in subsequent electrophoretic or chromatographic steps. The procedure can be applied both to the study of kinases present in crude protein mixtures and to the characterization of purified or partially purified enzymes. The in situ renaturation technique is generally applied to the detection of protein kinases either by autophosphorylation or by the phosphorylation of specific substrates included in the gel matrix. The experimental design determines the amount of protein that needs to be loaded onto the gel. While detecting protein kinase activities in crude cell extracts by autophosphorylation, the best results are obtained when large amounts of protein are loaded.

Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells.

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk− B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

The oxygen-substituted palmitic acid analogue, 13-oxypalmitic acid, inhibits Lck localization to lipid rafts and T cell signaling.

Palmitoylation of cysteines 3 and 5 is necessary for targeting Lck to lipid rafts and is needed for Lck function in T cell receptor (TCR) signaling. Point mutations of cysteines 3 and 5 result in a form of Lck that fails to associate with the plasma membrane, which limits the usefulness of this genetic approach to address the role of palmitoylation in the distribution of Lck within the plasma membrane. To circumvent this problem, we sought to identify a palmitic acid analogue that would enable plasma membrane association of Lck, but not facilitate its localization within lipid rafts. Here we examined the effects of the heteroatom-substituted analogue of palmitic acid, 13-oxypalmitic acid (13-OP), on Lck subcellular localization and function. 13-OP is similar in chain length to palmitic acid, but possesses reduced hydrophobicity. We found that treatment of cells with 13-OP inhibited incorporation of omega-[(125)I]iodopalmitate into Lck. 13-OP inhibited localization of Lck to lipid rafts without altering its membrane localization. Consistent with the dissociation of Lck from rafts, treatment with 13-OP abolished Lck association with the GPI-anchored protein, CD48, but not the transmembrane glycoprotein CD4. Jurkat T cells treated with 13-OP showed marked reduction in tyrosine phosphorylation and activation of mitogen-activated protein kinase upon TCR stimulation. In conclusion, the less hydrophobic analogue of palmitate, 13-OP, alters the normal acylation of Lck that provides Lck with the necessary hydrophobicity and tight packing order required for inclusion in lipid rafts.