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• W2028603863 abstract "Article1 May 1997free access Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets Atsushi Hirao Atsushi Hirao Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Isao Hamaguchi Isao Hamaguchi Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Toshio Suda Toshio Suda Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Naoto Yamaguchi Corresponding Author Naoto Yamaguchi Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Atsushi Hirao Atsushi Hirao Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Isao Hamaguchi Isao Hamaguchi Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Toshio Suda Toshio Suda Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Naoto Yamaguchi Corresponding Author Naoto Yamaguchi Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan Search for more papers by this author Author Information Atsushi Hirao1, Isao Hamaguchi1, Toshio Suda1 and Naoto Yamaguchi 1 1Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto, 860 Japan *E-mail: [email protected] The EMBO Journal (1997)16:2342-2351https://doi.org/10.1093/emboj/16.9.2342 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chk/Hyl is a recently isolated non-receptor tyrosine kinase with greatest homology to a ubiquitous negative regulator of Src family kinases, Csk. To understand the significance of co-expression of Chk and Csk in platelets, we examined the subcellular localization of each protein. Chk, but not Csk, was completely translocated from the Triton X-100-soluble to the Triton X-100-insoluble cytoskeletal fraction within 10 s of thrombin stimulation. Chk and Lyn, but not Csk and c-Src, co-fractionated in the higher density lysate fractions of resting platelets, with Chk being found to localize close to CD36 (membrane glycoprotein IV)-anchored Lyn. The kinase activity of co-fractionated Lyn was suppressed 3-fold. In vitro phosphorylation assays showed that Chk suppressed Lyn activity by phosphorylating its C-terminal negative regulatory tyrosine. Upon stimulation of platelets with thrombin, the rapid and complete translocation of Chk away from Lyn caused concomitant activation of Lyn. This activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. These results suggest that Chk, but not Csk, may function as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation. Introduction Src family protein tyrosine kinases play crucial roles in regulating proliferation and differentiation of multiple cell types, including hematopoietic cells (Bolen et al., 1992; Mustelin and Burn, 1993). The tyrosine kinase activity of Src family kinases is tightly regulated by tyrosine phosphorylation and dephosphorylation events (Cooper, 1990). The non-receptor-type tyrosine kinase C-terminal Src kinase (Csk), has been shown to phosphorylate the C–terminal negative regulatory tyrosine residue of Src family kinases and suppress their kinase activity (Okada and Nakagawa, 1989; Nada et al., 1991, 1993; Okada et al., 1991; Sabe et al., 1992; Bergman et al., 1992; Imamoto and Soriano, 1993). Hematopoietic consensus tyrosine-lacking kinase (Hyl) was cloned from the human megakaryocytic cell line UT–7 and found to possess greatest homology to Csk (Sakano et al., 1994). Like Csk, Hyl has Src homology 3 (SH3) and SH2 domains and lacks the consensus tyrosine phosphorylation and myristoylation sites found in Src family kinases. The Hyl and Csk genes are also structurally related, having the same intron–exon organization (Hamaguchi et al., 1994). cDNAs encoding proteins closely related to Csk and identical to Hyl, such as Matk, Ctk, Ntk, Lsk and Batk, have been reported (Bennett et al., 1994; Chow et al., 1994a; Klages et al., 1994; Kuo et al., 1994; McVicar et al., 1994). Since these cDNAs represent the mouse, rat and human homologs of the same gene, a new name for these kinases, Csk homologous kinase (Chk), has been proposed (D.W.McVicar, personal communication). Like Csk, Chk phosphorylates the negative regulatory tyrosine residues of Src family kinases in vitro and in a yeast co-expression system, suggesting that Chk may share functional properties with Csk (Chow et al., 1994a; Klages et al., 1994; Avraham et al., 1995). However, Csk is ubiquitously expressed, whereas Chk expression is restricted to hematopoietic cells and neuronal cells in the brain. The expression of both Chk and Csk in these cell types implies either functional redundancy or specific roles for both kinases. While recent studies indicate that Chk and Csk might differentially regulate the functions of Src family kinases (Chow et al., 1994b; Musso et al., 1994; Jhun et al., 1995), the function of Chk is still unknown. Activation of platelets with thrombin results in shape change, secretion of granular contents and aggregation. Platelets contain a number of non-receptor-type tyrosine kinases, including five Src family kinases (c-Src, c-Yes, Fyn, Lyn and Hck; Horak et al., 1990; Shattil and Brugge, 1991), Syk (Taniguchi et al., 1993) and FAK (Lipfert et al., 1992). Receptor-type tyrosine kinases have not been identified in platelets. During platelet activation, there is a rapid elevation of tyrosine-phosphorylated proteins due to activation of tyrosine kinases (Ferrel and Martin, 1988; Golden and Brugge, 1989; Nakamura and Yamamura, 1989; Dhar and Shukla, 1991; Clark et al., 1994; Schoenwaelder et al., 1994). c-Src and Syk have been shown to translocate from the cell membrane to the cytoskeletal fraction in activated platelets (Horvath et al., 1992; Oda et al., 1992; Clark and Brugge, 1993; Tohyama et al., 1994). Activation of downstream signaling cascades by these tyrosine kinases may require their association with the cytoskeleton. CD36 (glycoprotein IV, GP IV), one of the major platelet membrane glycoproteins, is physically associated with Lyn, Fyn and c-Yes (Huang et al., 1991). Interaction of CD36 with the fibrinogen-liganded form of integrin αIIbβ3 (GP IIb/IIIa) is hypothesized to stabilize platelet aggregation, leading to completion of the platelet activation process. In this report we show that Chk, unlike Csk, localizes with CD36-anchored Lyn and negatively regulates its activity in resting platelets. Upon thrombin stimulation the rapid and complete translocation of Chk triggers activation of CD36-anchored Lyn in co-operation with a protein tyrosine phosphatase to dephosphorylate its negative regulatory tyrosine residue. Our findings suggest that Chk, but not Csk, plays an important role in the regulation of kinase activity of CD36-anchored Lyn in thrombin-stimulated platelets. Results Detection of Chk in platelets using monoclonal antibodies Using a GST–Chk fusion protein and a synthetic peptide of the C-terminal region of Chk as antigens, two monoclonal antibodies (13G2 and 18E12) and an affinity-purified polyclonal antibody (C-2930) against Chk were generated. To verify the specificity, expression of Chk and FLAG epitope-tagged Chk (Chk–FLAG) in COS-7 cells was examined. A single protein of 57 kDa was immunoprecipitated with 13G2 or 18E12 from the lysate of [35S]Met-labeled COS-7 cells transfected with Chk (Figure 1A). The immunoprecipitate with 13G2 or 18E12 from the lysate of COS-7 cells transfected with Chk–FLAG was detected by both C-2930 and anti-FLAG (Figure 1B). 13G2 also detected Chk in the lysate of COS-7 cells transfected with Chk by Western blotting (Figure 1C). These results confirm that these monoclonal antibodies specifically recognize Chk. Figure 1.Detection of Chk protein. (A) Fluorogram of SDS–PAGE of immunoprecipitates with two monoclonal antibodies, 13G2 and 18E12, from metabolically labeled COS-7 cells transfected with Chk or vector alone. (B) Western blots of immunoprecipitates from COS-7 cells transfected with Chk–FLAG or vector alone probed with anti-Chk (Po: C-2930) or anti-FLAG. IgG(H), heavy chain of immunoglobulin G. (C) Western blot of Triton X-100-soluble lysates from various cells, probed with anti-Chk (13G2). Download figure Download PowerPoint Next, we examined expression of Chk in several human cell lines by Western blotting. Figure 1C shows that Chk was detected at 57 kDa in UT-7 cells (megakaryocytic) and CMK cells (megakaryoblastic) but not in U937 cells (monocytic), consistent with previous observations of Chk mRNA expression (Bennett et al., 1994; Sakano et al., 1994). Notably, Chk is expressed in human platelets (Figure 1C) Translocation of Chk to the cytoskeleton in thrombin-activated platelets Since c-Src and Syk are translocated from the Triton X–100-soluble fraction to the Triton X-100-insoluble fraction (i.e. the cytoskeleton) in activated platelets, we investigated whether Chk was translocated to the cytoskeleton. After stimulation with thrombin for 15 s, Chk was completely translocated to the Triton X-100-insoluble fraction and then gradually degraded (Figure 2A). When platelets were incubated with inactivated thrombin treated with D–phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK), translocation of Chk to the Triton X–100-insoluble fraction was not observed (data not shown). Figure 2.Distribution of Chk, c-Src and Csk during thrombin stimulation. (Left panels) Western blots of Triton X-100-soluble and Triton X-100-insoluble fractions of platelets stimulated for the indicated times (s) with thrombin probed with anti-Chk (13G2) (A), anti-Src (B) and anti-Csk (C). (Right panels) densitometric quantification of the amounts (%) of kinases in Triton X-100-soluble (open circles) and Triton X-100-insoluble (closed circles) fractions. Download figure Download PowerPoint On the other hand, c-Src was translocated to the Triton X-100-insoluble fraction at a relatively slow rate during thrombin stimulation and only 20% of c-Src was translocated (Figure 2B), consistent with previous observations (Clark and Brugge, 1993). Csk was also detected in the Triton X-100-soluble fraction (Figure 2C). Interestingly, <3% of Csk was translocated to the Triton X-100-insoluble fraction upon thrombin stimulation, with dynamics similar to those of c-Src (Figure 2C), implying that Chk and Csk may play different roles in platelet activation. Kinase activities of Chk during thrombin stimulation To examine whether the translocation of Chk affected its kinase activity, immune complex kinase assays were performed. Resting and stimulated platelets were solubilized with RIPA buffer to recover Chk from both the Triton X-100-soluble and Triton X-100-insoluble fractions. Figure 3A shows that Chk immunoprecipitated from resting and stimulated platelets had nearly the same kinase activity on an exogenous substrate, poly(Glu,Tyr). We next examined the activity of Chk with c-Src as substrate. Autophosphorylation of c-Src was inhibited by treatment with an ATP analog, p-fluoro-sulfonylbenzoyl 5′-adenosine (FSBA), which is known to inactivate c-Src by reacting with Lys295 (Figure 3B, lanes 1 and 2); Kamps et al., 1984; Okada and Nakagawa, 1989). The inactivated c-Src was phosphorylated by Chk immunoprecipitated from resting or stimulated platelets (lanes 3–6). The relative specific activities of Chk immunoprecipitated from resting and stimulated platelets were estimated to be nearly the same by normalizing the Chk activity to the amounts of Chk shown in the lower panel. Furthermore, the relative specific activities of Csk immunoprecipitated from resting and stimulated platelets were also estimated to be the same (data not shown). These results suggest that thrombin stimulation does not affect the activities of Chk and Csk. Figure 3.Kinase activities of Chk during thrombin stimulation. (A) In vitro kinase assays of immunoprecipitates with anti-Chk (13G2) from platelets stimulated with thrombin for the indicated times. Kinase activities are expressed as the values [means ± SE of three different experiments (%)] relative to the specific activity of Chk in the unstimulated sample after normalizing the incorporation of 32PO4 into poly(Glu,Tyr) for each level of Chk protein. (B) (Upper panel) autoradiogram of in vitro phosphorylation of c-Src (lane 1) and of FSBA-treated c-Src (lanes 2–6) by addition of immunoprecipitates with MOPC21 as a control antibody (lanes 3 and 5) or with anti-Chk (13G2) (lanes 4 and 6) from platelets incubated for 30 s with (lanes 5 and 6) or without (lanes 3 and 4) thrombin. (Lower panel) Western blot of the added immunoprecipitates probed with anti-Chk (13G2). Download figure Download PowerPoint Plasma membrane anchorage of Chk with a protein complex Although Chk does not possess any known membrane anchoring motifs, in resting platelets >90% of Chk was localized to the membrane fraction, with the remainder in the cytosol fraction (Figure 4A). In sharp contrast, most Csk was localized to the cytosol fraction. c-Src and Lyn were localized to the membrane fraction, as expected due to their post-translational lipid modification. Figure 4B shows fractionation of a 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate (CHAPS) lysate obtained from resting platelets on density gradients. All Chk and a large amount of Lyn co-fractionated in the higher density lysate fractions (fraction 5 and later fractions, >200 kDa as judged from calibration with molecular markers), whereas all Csk and ∼50% of c-Src were detected primarily as monomers. Moreover, cell surface biotinylation of resting platelets revealed 68, 58 and 52 kDa proteins co-immunoprecipitating with Chk, while no biotinylated proteins were co-immunoprecipitated with Csk (Figure 4C). These results suggest that Chk, but not Csk, forms a complex and is physically associated with cell surface membrane proteins. Figure 4.Localization of Chk in resting platelets. Western blots of subcellular fractions (A) and of lysates fractionated on sucrose density gradients (B) probed with anti-Chk (13G2), anti-Csk, anti-Src and anti-Lyn. (C) Western blots of immunoprecipitates with MOPC21 as a control, anti-Chk (13G2) or anti-Csk from surface-biotinylated platelets probed with HRP-conjugated streptavidin (upper panel) or anti-Chk (13G2) and anti-Csk (lower panels). Chk, 57 kDa; Csk, 50 kDa; c-Src, 60 kDa; Lyn, 53 and 56 kDa. Download figure Download PowerPoint Association of Chk with a complex of CD36 including Lyn CD36 is reported to associate with Lyn (Huang et al., 1991). In addition, the distributions of Chk and Lyn overlap with each other (Figure 4B) and the patterns of Chk and CD36 were nearly the same on density gradients (fraction 5 and later fractions; data not shown). We therefore examined whether these kinases physically associated with CD36 in resting platelets. Figure 5A shows that after chemical crosslinking Chk was detected in an immunoprecipitate with anti-CD36. Without chemical crosslinking, an association of Chk with the immune complex of CD36 was not detected (data not shown). The immune complex of CD36 also contained Lyn, but not c–Src (Figure 5A), consistent with previous observations (Huang et al., 1991). In contrast to Chk, Csk was not detected in the immune complex of CD36 with (Figure 5A) or without chemical crosslinking (data not shown). Figure 5.Association of Chk with a CD36 complex. (A) Western blots of immunoprecipitates with MOPC21 as a control and anti-CD36 from platelets chemically crosslinked with DSP and (B) Western blots of platelet membrane vesicles purified using anti-CD36, anti-αIIbβ3, anti-GPIb or MOPC21 as a control probed with anti-Chk (13G2), anti-CD36, anti-Lyn, anti-Csk, anti-Src, anti-β3 and anti-GPIb, as indicated. Download figure Download PowerPoint We then attempted to characterize further the relationship between Chk and CD36 (Figure 5B) membrane vesicles were prepared from sonicated platelets and affinity purified using anti-CD36, anti-αIIbβ3 or anti-GPIb. Vesicles predominantly expressing CD36 or GPIb were purified. αIIbβ3 molecules were present in the CD36- and GPIb-expressing vesicles as well as in the vesicles purified with anti-αIIbβ3. Chk was detected in the CD36-expressing vesicles but not in the GPIb- or αIIbβ3-expressing vesicles, whereas Lyn and c-Src were found in each membrane vesicle fraction. In contrast to Chk, Csk was not detected in any membrane vesicle fraction (data not shown). These results suggest that Chk, but not Csk, is localized with CD36-anchored Lyn in resting platelets. Effect of Chk phosphorylation on kinase activity of Lyn To examine whether Lyn was phosphorylated and negatively regulated by Chk, Lyn immunoprecipitates were subjected to in vitro kinase assays. Figure 6A shows that autophosphorylation of Lyn was inhibited by treatment with FSBA (lanes 1 and 2) and that the inactivated Lyn was phosphorylated by Chk (lane 3). Thus, Lyn, as well as c-Src (Figure 3A), was found to be a substrate for Chk. Next, purified Lyn was first phosphorylated by various amounts of purified Chk in the presence of unlabeled ATP and then the activity of Lyn was measured. Figure 6B shows that the extent of phosphorylation of enolase and of autophosphorylation of Lyn was reduced by increasing the amount of Chk added, indicating that phosphorylation of Lyn by Chk results in a decrease in the kinase activity of Lyn. Figure 6.Effect of Chk phosphorylation on kinase activity of Lyn. (A) Autoradiogram of in vitro phosphorylation of Lyn immunoprecipitates from platelets. Lane 1, autophosphorylation of Lyn; lane 2, autophosphorylation of FSBA-treated Lyn; lane 3, phosphorylation of FSBA-treated Lyn by addition of purified Chk–FLAG. (B) Autoradiogram of in vitro Lyn phosphorylation regulated by Chk. The purified Lyn was first incubated with various amounts of purified Chk–FLAG (lane 1, 0 μl; lane 2, 1 μl; lane 3, 5 μl; lane 4, 10 μl) in the presence of unlabeled ATP (2 μM) for 15 min and further incubated with acid-denatured enolase in the presence of 1.5 μM [γ-32P]ATP for 60 min. Lyn kinase activities were determined by measuring the incorporation of 32PO4 into enolase. The values are expressed as percentages of the control in the absence of Chk–FLAG. (C) Autoradiogram of CNBr-cleaved fragments of Lyn. Upper panel: a schematic representation of the CNBr cleavage sites in Lyn. The 8 and 4 kDa fragments contain the autophosphorylation site (Tyr397) and the negative regulatory site (Tyr508) respectively. Lower panel: Lyn proteins were purified and analyzed by cleavage with CNBr from in vivo 32PO4-labeled COS-7 cells co-expressing either Lyn and Chk–FLAG (left lane) or Lyn and Chk–FLAG (K262R) (right lane). Download figure Download PowerPoint Since negative regulation of Lyn by Csk is due to phosphorylation at the C-terminal tyrosine residue, Tyr508, of Lyn (Okada et al., 1991), we investigated whether Chk could phosphorylate this negative regulatory tyrosine residue on Lyn. COS-7 cells co-expressing Lyn and Chk were in vivo labeled for 3 h with H332PO4. Phosphorylated Lyn was purified, cleaved with cyanogen bromide (CNBr) and analyzed by SDS–PAGE. Figure 6C shows the major phosphorylated bands of the 8 kDa fragment containing Tyr397 and the 4 kDa fragment containing Tyr508 (left lane in lower panel). Co-expression of Lyn and kinase-inactive Chk resulted in a major phosphorylated band of the 8 kDa fragment containing Tyr397 (right lane in lower panel). This analysis of CNBr cleavage products indicated that Chk-dependent phosphorylation of Lyn occurs on the 4 kDa fragment containing Tyr508. These results suggest that Chk suppresses Lyn kinase activity by phosphorylation of its negative regulatory tyrosine residue. Functional association of Chk with CD36-anchored Lyn To examine the effect of Chk on the kinase activity of Lyn in resting platelets, two Lyn immunoprecipitates were subjected to an in vitro autophosphorylation assay, one that co-fractionated with Chk (Figure 4B, fraction 13) and one that did not (fraction 4). Figure 7A shows that autophosphorylation of Lyn immunoprecipitated from the fraction containing Chk was suppressed. The level of activity of this Lyn species was estimated to be ∼3-fold lower (right lane) than that of the other species of Lyn in the fraction lacking Chk (left lane) when activity was normalized to the amount of Lyn in each sample. This result suggests that Chk may negatively regulate the kinase activity of Lyn associated with CD36 in resting platelets. Figure 7.Functional association of Chk with CD36-anchored Lyn. (A) Autoradiogram of in vitro autophosphorylation of Lyn immunoprecipitated from density gradient fractions 4 and 13 shown in Figure 4B (upper panel) and the amounts of Lyn immunoprecipitates, blotted with anti-Lyn (lower panel). (B) Western blots of the Triton X-100-soluble (left panel) and Triton X-100-insoluble (right panel) fractions of platelets stimulated with thrombin for the indicated times probed with anti-Chk (13G2), anti-CD36 and anti-Lyn. (C) In vitro kinase assays of Lyn immunoprecipitates from the Triton-soluble fractions of platelets stimulated with thrombin for the indicated times. Kinase activities are expressed as values relative to the specific activity of Lyn in the unstimulated sample after normalizing for incorporation of 32PO4 into enolase for each level of Lyn protein. The graph represents results from four different experiments. (D) Autoradiograms of in vivo phosphorylation of the negative regulatory sites of Lyn and c-Src present in the Triton-soluble fractions upon stimulation. After in vivo labeling, stimulation and purification of Lyn and c-Src, the proteins were cleaved with CNBr. Upper panels show phosphorylation states of the resulting 4 kDa fragments of Lyn (left) and c-Src (right) from platelets stimulated with thrombin for 0 (lanes 1 and 4), 10 (lanes 2 and 5) or 60 s (lanes 3 and 6). Incorporation of 32PO4 into the 4 kDa fragments was quantitated. In vitro kinase activities of Lyn and c-Src immunoprecipitates from the Triton-soluble fractions of stimulated platelets (0, 10 or 60 s) are expressed as described above. The graphs represent results from (C) for Lyn and from three different experiments for c-Src. Download figure Download PowerPoint Figure 7B shows that upon thrombin stimulation CD36 was translocated from the Triton X-100-soluble to Triton X-100-insoluble fraction with dynamics similar to those of Lyn, indicating that Lyn continues to anchor CD36 during thrombin stimulation. Although progressive translocation of both CD36 and Lyn from the Triton X–100-soluble to Triton X-100-insoluble fraction was observed, Chk was completely translocated within 10 s of thrombin stimulation (Figure 7B). This result suggests that translocation of Chk to the cytoskeleton may be completed rapidly, before the beginning of translocation of most CD36-anchored Lyn. It is therefore possible that rapid translocation of Chk releases CD36-anchored Lyn from negative regulation. To examine whether Lyn was activated before its translocation to the cytoskeleton, Lyn was immunoprecipitated from the Triton X-100-soluble fraction and in vitro kinase assays were performed with enolase. An increase in the kinase activity of Lyn was detected at 10 s after thrombin stimulation (Figure 7C). The increase was 1.7- to 2.2-fold (n = 4), peaked at 30–60 s after thrombin stimulation and was then sustained, with a slight reduction (6–18%) after 120 s stimulation. This result suggests that activation of Lyn occurs before its translocation to the cytoskeleton. Although Chk was found to suppress Lyn kinase activity by phosphorylation of its C-terminal negative regulatory tyrosine residue (Figure 6), the rapid translocation of Chk away from Lyn may not be sufficient for activation of Lyn. Dephosphorylation of the negative regulatory tyrosine residue of Lyn by a protein tyrosine phosphatase may be also required for activation of Lyn. We then examined in vivo phosphorylation states of the negative regulatory tyrosine residue of Lyn during thrombin stimulation. In vivo 32PO4-labeled platelets were stimulated with thrombin for 0, 10 or 60 s and phosphorylated Lyn and c-Src were purified from the Triton X-100-soluble fraction before their translocation to the Triton X–100-insoluble fraction. After confirming that each sample contained comparable amounts of purified Lyn or c-Src, the phosphorylation states of Lyn and c-Src were analyzed by CNBr cleavage. Figure 7D shows that a decrease in phosphorylation of the 4 kDa fragment of Lyn containing Tyr508 occurred at 10 s after thrombin stimulation (upper left). The level of phosphorylation was further decreased at 60 s after stimulation. On the other hand, Figure 7D also shows different phosphorylation states between Lyn and c-Src at the negative regulatory tyrosine residue during thrombin stimulation. A transient and slight decrease in phosphorylation of the 4 kDa fragment containing the negative regulatory tyrosine residue, Tyr530, of c-Src was observed at 10 s after stimulation (upper right), consistent with previous observations (Clark and Brugge, 1993). These results probably reflect an association of Lyn, but not c–Src, with CD36 (Figure 5A). Furthermore, upon thrombin stimulation the extent of decrease in phosphorylation of the negative regulatory tyrosine residue corresponded to the level of kinase activity of Lyn and c-Src (compare upper panels with lower panels in Figure 7D. Therefore, these results suggest that dephosphorylation of the negative regulatory tyrosine residue by a protein tyrosine phosphatase in a Lyn-specific manner gives rise to activation of Lyn before its translocation to the cytoskeleton. Discussion In this report we demonstrate co-expression of Chk and Csk in platelets and the significance of the unique subcellular localization of Chk in thrombin-induced platelet activation. Subcellular localizations of Chk and Csk We generated monoclonal antibodies against Chk (Figure 1) and found that most of the Chk protein is localized in the Triton X-100-soluble membrane fraction (Figure 4A). Since Chk has been shown to localize mainly to the cytoplasm and appreciably to the detergent-insoluble fraction of several cell lines, but not to the membrane fraction (Bennett et al., 1994; Chow et al., 1994b), the membrane localization of Chk in platelets is quite unique. In contrast, the finding that most Csk is present in the cytosol (Figure 4A) agrees with previous data that most Csk protein is localized in the cytoplasm of fibroblastic cells, although a small amount of Csk is concentrated in the membrane fraction, particularly in adhesion plaques (Nada et al., 1991; Sabe et al., 1992; Howell and Cooper, 1994; Bergman et al., 1995). Although Csk and Chk are homologous proteins, Chk contains a unique 41 amino acid N-terminus and the Chk SH3 and SH2 domains show only 30 and 59% amino acid identity respectively to Csk. It is possible that the differential binding to membrane proteins exhibited by Chk and Csk may be the result of these structural differences. In fact, co-immunoprecipitation analysis showed a physical association of Chk with 68, 58 and 52 kDa biotinylated cell surface proteins in lysis buffer containing 1% Triton X-100 (Figure 4C). This finding indicates an interaction of Chk with the cytoplasmic portion of a surface membrane protein(s). Additionally, density gradient analysis, showing that Chk, unlike Csk, is present in the higher density fractions (>200 kDa; Figure 4B supports complex formation by Chk and membrane proteins. However, further studies are required to identify the molecules associated with Chk. Moreover, it is reported that platelets contain five Src family kinases, i.e. c-Src, Lyn, Fyn, c-Yes and Hck, three of which, Lyn, Fyn, and c-Yes, stably interact with one of the major platelet membrane glycoproteins, CD36/GPIV (Huang et al., 1991). In addition to the result that Lyn, unlike Csk and c-Src, is also located in the higher density fractions (Figure 4B), our immunoprecipitation study revealed that a complex of CD36 with Lyn is chemically crosslinked with Chk (Figure 5A). In the absence of a chemical crosslinker, physical association could not be detected between Chk and a complex of CD36 with Lyn. Nonetheless, Chk and CD36–Lyn may be adjacent to and interact with each other because the spacer arm length of dithiobis(suc" @default.
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• W2028603863 date "1997-05-01" @default.
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• W2028603863 title "Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets" @default.
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