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The Journal of Immunology, 2002, 169: 2813-2817.
Copyright © 2002 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Fyn Is Essential for Tyrosine Phosphorylation of Csk-Binding Protein/Phosphoprotein Associated with Glycolipid-Enriched Microdomains in Lipid Rafts in Resting T Cells1

Koubun Yasuda2,*, Masakazu Nagafuku2,{dagger}, Takaki Shima{ddagger}, Masato Okada{ddagger}, Takeshi Yagi§, Takenao Yamada{dagger}, Yasuko Minaki{dagger}, Akiko Kato{dagger}, Shizue Tani-ichi{dagger}, Toshiyuki Hamaoka* and Atsushi Kosugi3,{dagger},¶

* Department of Oncogenesis, Graduate School of Medicine, {dagger} Faculty of Medicine, School of Allied Health Sciences, {ddagger} Department of Oncogene Research, Research Institute for Microbial Diseases, § Division of Molecular Genetics, Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan; and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In resting T cells, Csk is constitutively localized in lipid rafts by virtue of interaction with a phosphorylated adaptor protein, Csk-binding protein (Cbp)/phosphoprotein associated with glycolipid-enriched microdomains, and sets an activation threshold in TCR signaling. In this study, we examined a kinase responsible for Cbp phosphorylation in T cell membrane rafts. By analyzing T cells from Fyn-/- mice, we clearly demonstrated that Fyn, but not Lck, has its kinase activity in membrane rafts, and plays a critical role in Cbp phosphorylation, Cbp-Csk interaction, and Csk kinase activity. Naive CD44lowCD62 ligandhigh T cells were substantially reduced in Fyn-/- mice, presumably due to the inhibition of Cbp phosphorylation. Thus, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts by phosphorylating Cbp. Csk recruited to rafts would then be activated and inhibit the kinase activity of Lck to keep resting T cells in a quiescent state. Our results elucidate a negative regulatory role for Fyn in proximal TCR signaling in lipid rafts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One of the earliest biochemical events after TCR engagement is the tyrosine phosphorylation of several proteins by protein tyrosine kinases (PTKs).4 It has been demonstrated that the initial TCR signal is mediated by the activation of the Src family PTKs Lck and Fyn (1, 2, 3). The Src family PTKs are negatively regulated by phosphorylation of a conserved C-terminal tyrosine residue by Csk (4, 5). Csk is a cytoplasmic PTK consisting of an Src homology domain 3, an Src homology domain 2, and a kinase domain similar to the Src family PTKs. However, because it lacks an N-terminal acylation signal and a C-terminal tyrosine, the regulatory mechanism of Csk itself has remained unknown. Recently, a new adaptor protein capable of recruiting Csk to the plasma membrane was identified. This protein called Csk-binding protein (Cbp; 6) or phosphoprotein associated with glycolipid-enriched microdomains (PAG; 7) is exclusively localized in lipid rafts and has the capacity to bind Csk by virtue of its phosphorylation on Tyr314. Lipid rafts, specialized compartments in the plasma membrane, are known to act as signaling platforms that facilitate intramolecular association and the propagation of signal transduction cascades in many cell types including lymphocytes (8). Csk is associated with rafts via an interaction with phosphorylated Cbp/PAG in resting T cells, and may regulate an activation threshold of T cells through the inhibition of the Src family PTKs (9).

Although Cbp/PAG thus functions as a negative regulator of the Src family PTKs by its interaction with Csk, phosphorylation of Cbp/PAG itself could be controlled by the Src family PTKs (6, 7). Coexpression of c-Src and Cbp in 293T cells resulted in Cbp phosphorylation. PAG served as a substrate for Lck and Fyn, but not for ZAP-70 and/or Syk in COS cells expressing PAG and various PTKs; and selective inhibitors for the Src family PTKs, PP1 and PP2, suppressed Cbp/PAG phosphorylation both in vivo and in vitro. However, it remains unknown which kinase, Lck or Fyn, is involved in physiological regulation for Cbp phosphorylation in T cells. Moreover, a molecular mechanism for activation and inactivation of the Src family PTKs in lipid rafts in TCR signaling is also still elusive. In this study, we demonstrated that Fyn, but not Lck, has a high catalytic activity in rafts and is responsible for Cbp phosphorylation in T cells at the resting stage. Our results clearly demonstrate that Fyn is critical for Cbp-Csk interaction in proximal TCR signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The Fyn-/- mice were generated as previously described (10). The heterozygous Fyn+/- mice were back-crossed with C57BL/6 mice for six generations, and the B6-Fyn+/- mice were then intercrossed to produce homozygous Fyn-/- B6 mice. Mice were analyzed for the fyn gene genotype by PCR (data not shown).

Abs and reagents

The following Abs were used: MOL 171 (11), anti-Lck mAb; OKT3 (American Type Culture Collection, Manassas, VA), anti-human CD3{epsilon} mAb; anti-Cbp and anti-Csk polyclonal Abs (6); antitransferrin receptor mAb (Zymed Laboratories, San Francisco, CA); anti-Fyn mAb (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Lck Ab (Upstate Biotechnology, Lake Placid, NY); anti-phospho-Lck (Tyr505) Ab (Cell Signaling Technology, Beverly, MA); PY20, anti-phosphotyrosine mAb (Transduction Laboratories, Lexington, KY); and goat anti-hamster IgG (Cappel, Aurora, OH). Dye-labeled Abs used for flow cytometry were purchased from BD PharMingen (San Diego, CA). Cholera toxin B-HRP, enolase, and a random polymer of glutamate and tyrosine (poly EY) were purchased from Sigma-Aldrich (St. Louis, MO).

Isolation of a raft fraction, immunoprecipitation, and immunoblotting analysis

Isolation of a raft fraction, immunoprecipitation, and immunoblotting analysis were performed as previously described (11).

In vitro kinase assays

Lck or Fyn immunoprecipitates (IPs) were washed twice with washing buffer (MES-buffered saline (25 mM MES (pH 6.5), 150 mM NaCl) with 0.1% Triton X (TX)-100) and twice with kinase buffer (KB; 50 mM HEPES (pH 7.4), 10 mM MgCl2, 5 mM MnCl2, and 0.5 mM sodium orthovanadate). The IPs suspended in KB with 1 µg of acid-denatured enolase and 10 µCi [{gamma}-32P]ATP were incubated at 30°C for 15 min and stopped by addition of 5x SDS-PAGE sample buffer. The enolase and IPs were separated by SDS-PAGE and detected by autoradiography. The relative amount of Lck or Fyn present in each sample was detected by immunoblotting. The kinase activity of Csk was measured by [32P]O4 labeling of poly EY as a substrate. Cbp IPs was suspended in KB with 4 µg of poly EY and 10 µCi [{gamma}-32P]ATP. To block Cbp-Csk interaction, Cbp-4 (ISAMYSSVNK) or Cbp-2 (EDCLYETVKE) phosphopeptides were added to cell lysates before immunoprecipitation with anti-Cbp Abs as previously described (12). The activity was visualized and quantified using a BAS-1500 Bioimage Analyzer (Fuji Photo Film, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The kinase activities of Lck and Fyn are differentially localized in T cell plasma membranes

To investigate the localization of Lck and Fyn kinase activities in T cells, we isolated the raft fraction from Jurkat cells using a sucrose gradient. Successful fractionation was confirmed by the localization of a raft and a nonraft marker, GM1, and transferrin receptor, respectively (Fig. 1GoA). In vitro kinase assays were performed using Lck or Fyn immunoprecipitated from the raft and the nonraft fractions. The kinase activity was measured by [32P]O4 labeling of enolase, a specific substrate for the Src family kinases, and the amount of Lck or Fyn present in each fraction was measured by immunoblotting (Fig. 1GoB). Intriguingly, although the amount of Lck was highly concentrated in the raft fraction, Lck kinase activity was predominantly detected in the nonraft fraction. This is consistent with previous findings by Rodgers and Rose (13). TCR stimulation did not increase Lck kinase activity nor change its localization among the fractions. In contrast to Lck, Fyn kinase activity was highly enriched in the raft fraction before and after TCR stimulation. The differential localization of Lck and Fyn was not changed at the later time point, although the activities per se were decreased 60 min after TCR cross-linking (data not shown).



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  		FIGURE 1. The kinase activities of Lck and Fyn are differentially localized in T cell plasma membranes. A, Jurkat cells and T cells from normal B6 mice (Spl, splenic T cell; LN, lymph node T cell; and Thy, thymocyte) were lysed with MES-buffered saline containing 1% TX-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot from fraction 4 (raft, the raft fraction) or from fraction 12 (TXS, the TX-soluble fraction) was electrophoresed and tested for their levels of GM1 and transferrin receptor (TfR) by immunoblotting analysis. B, In vitro kinase assays of Lck or Fyn from the raft and the TXS fractions. Jurkat cells were either left untreated (-) or were stimulated with OKT3 for 2 min (+) before the isolation of the raft and the TXS fractions. In vitro kinase assays were performed using Lck or Fyn from each membrane fraction after immunoprecipitation with anti-Lck or anti-Fyn Abs, respectively. Enolase was used as an exogenous substrate. The amount of Lck or Fyn in each fraction was measured by immunoblotting. C, In vitro kinase assays of Lck or Fyn in the raft and the TXS fractions from mouse T cells.

 
To examine whether Lck and Fyn localized in rafts also display different levels of the kinase activity in freshly isolated T cells, we next prepared the raft and the nonraft fractions from mouse thymocytes, splenic, and lymph node T cells (Fig. 1GoC). Although the percentage of Lck that resided in the raft fraction varied between each cell type, it was obvious that most of Lck kinase activity was localized in the nonraft fractions in thymocytes and peripheral T cells. However, again Fyn kinase activity was clearly detected in the raft fractions. Because the amount of Fyn in the nonraft fractions was small but it had a considerable level of the kinase activity, the relative activity per molecule in the nonraft fraction could be higher than that in the raft fraction. Nonetheless, Fyn differed from Lck in that it had substantial kinase activity in the raft fraction. Stimulation of the TCR did not detectably alter Lck and Fyn kinase activities in thymocytes and peripheral T cells (data not shown).

Together, in contrast to the low activity of Lck, Fyn has a catalytic activity in rafts both in Jurkat and normal T cells even before T cell activation. Successful detection for Fyn kinase activity using the raft fraction effectively eliminates the possibility that lack of Lck kinase activity in the raft fraction was due to general disruption of enzymatic activity in this fraction caused by equilibrium density gradient centrifugation.

Fyn is essential for Cbp/PAG phosphorylation in resting T cells

The substantial kinase activity of Fyn in rafts from Jurkat, thymocytes, and peripheral T cells before activation prompted us to examine a specific role for this kinase in raft-mediated T cell activation. Recently, a new adaptor molecule, Cbp, which exerts an important function in Src family PTK signaling pathways, has been identified. Upon phosphorylation, Cbp, which is constitutively localized in rafts, can recruit Csk to rafts. We next investigated which kinase is responsible for Cbp phosphorylation in T cell membranes. Because raft-associated Fyn, but not Lck, had a catalytic activity in T cells before activation, we speculated that Cbp is phosphorylated by Fyn in resting T cells. To address this point, we used T cells from Fyn-/- mice. The phosphorylation level of Cbp was investigated using thymocytes and peripheral T cells from Fyn-/- mice. As shown in Fig. 2Go, A and B, Cbp phosphorylation was greatly reduced in T cells from Fyn-/- mice. In three independent experiments, the levels of Cbp phosphorylation in peripheral T cells and thymocytes from Fyn-/- mice were reduced to 12 and 18%, respectively, compared with those from normal mice. In contrast, Cbp expression was comparable between T cells from normal and Fyn-/- mice, although the level of Cbp expression seemed to vary in T cells from individual Fyn-/- mice. Because Cbp phosphorylation was not completely inhibited in Fyn-/- T cells, it is likely that kinases other than Fyn are also involved in Cbp phosphorylation. Nonetheless, the results using Fyn-/- T cells clearly demonstrated that Fyn is predominantly responsible for the phosphorylation of Cbp in resting T cells.



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  		FIGURE 2. Inhibition of Cbp phosphorylation in Fyn-/- T cells. A, Lymph node T cells and thymocytes from normal B6 or Fyn-/- mice were lysed, immunoprecipitated with anti-Cbp, and immunoblotted with anti-phosphotyrosine (PY) or anti-Cbp Abs. B, The graph shows the average of three experiments where the percentage of intensity of phosphorylated and total Cbp in Fyn-/- T cells against those in normal B6 T cells was calculated. Each experiment was done as described in A.

 
The amount and the kinase activity of Csk associated with Cbp in Fyn-/- mice

We next investigated whether decreased Cbp phosphorylation may alter Cbp-Csk interaction and Csk activity in Fyn-/- T cells. Thymocytes and peripheral T cells from normal and Fyn-/- mice were solubilized with lysis buffer containing n-octyl-{beta}-D-glucoside, immunoprecipitated with anti-Cbp, and immunoblotted with anti-Csk. The amounts of Csk associated with Cbp in thymocytes and peripheral T cells from Fyn-/- mice were decreased to 29 and 31%, respectively, of those from normal mice on the average from three experiments (Fig. 3GoA). To determine whether this reduction in Cbp-Csk interaction resulted in a decrease in Csk activity, we examined Csk activity in in vitro kinase assays using poly EY as substrate. As shown in Fig. 3GoB, Cbp IPs from normal thymocytes were capable of phosphorylating poly EY efficiently, and this phosphorylation was almost completely blocked by the addition of Cbp-4 phosphopeptide which contains Tyr314 and is known to compete with Cbp-Csk interaction (12). Another phosphopeptide, Cbp-2, which cannot interfere with the binding of Cbp to Csk was not able to inhibit the phosphorylation of poly EY. These results clearly demonstrate that the phosphorylation of poly EY in Cbp IPs was specifically attributable to the kinase activity of Csk. The kinase activity of Cbp-associated Csk in Fyn-/- thymocytes was decreased to 50% of that from normal thymocytes (Fig. 3GoB, lines 1 and 4). We observed a similar inhibition of Csk activity in Fyn-/- peripheral T cells (data not shown). Thus, the inhibition of Cbp phosphorylation in Fyn-/- T cells impaired Cbp-Csk interaction and the kinase activity of Csk.



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  		FIGURE 3. The amount and the kinase activity of Csk associated with Cbp, and Tyr505 phosphorylation of Lck in Fyn-/- T cells. A, The amount of Csk bound to Cbp. Lymph node T cells and thymocytes from normal B6 or Fyn-/- mice were lysed, immunoprecipitated with anti-Cbp, and immunoblotted with anti-Csk or anti-Cbp Abs. The graph shows the average of three experiments where the percentage of intensity of Csk bound to Cbp and total Cbp in Fyn-/- T cells against those in normal B6 T cells was calculated. B, Cbp IPs from thymocytes of normal B6 or Fyn-/- mice was assessed for Csk kinase activity using poly EY as a substrate. Lysates of thymocytes were immunoprecipitated with anti-Cbp Abs in the presence or absence of Cbp phosphopeptides as indicated. Cbp-4 that contains Tyr314 is able to inhibit Cbp-Csk interaction, while Cbp-2 is not. Csk was purified from infected insect cells as described previously (12 ). C, Tyr505 phosphorylation and the total amount of Lck in the raft fraction from normal B6 or Fyn-/- thymocytes were analyzed by anti-phospho-Lck (Y505) and anti-Lck blotting, respectively.

 
Reduced phosphorylation on Tyr505 of Lck in the raft fraction from Fyn-/- T cells

The kinase activity of Lck is negatively regulated by phosphorylation of a C-terminal residue (Tyr505 in Lck) by Csk. We next analyzed the phosphorylation level of Lck Tyr505 in Fyn-/- T cells using a phosphospecific Ab to Tyr505 of Lck. We especially focused on Lck Tyr505 phosphorylation in the raft fraction because we have observed that phosphorylation of Tyr505 was predominantly detected in the raft fraction (data not shown). Although the amount of Lck was comparable between normal and Fyn-/- thymocytes, Tyr505 phosphorylation in the raft fraction was clearly decreased in Fyn-/- thymocytes (Fig. 3GoC). This result indicates that Fyn promotes phosphorylation of the negative regulatory tyrosine of Lck by way of phosphorylating Cbp and recruiting Csk in lipid rafts.

Naive phenotype T cells decrease in Fyn-/- mice

Because the data presented above demonstrated that Fyn regulates Cbp-Csk interaction and Csk activity in resting T cells, we next asked whether this regulation influences the maintenance of naive T cells. Naive and memory T cells can be defined by the expression of surface markers, CD44 and CD62 ligand (CD62L; L-selectin; Ref. 14). As shown in Fig. 4Go, naive CD44lowCD62Lhigh T cells were substantially reduced in both the CD4+ and CD8+ T cell populations from Fyn-/- mice compared with those from normal mice. This result suggests that the regulation of Cbp-Csk interaction by Fyn is important for keeping peripheral T cells in a resting state in vivo.



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  		FIGURE 4. Expression of CD44 and CD62L on the surface of Fyn-/- T cells. Spleen cells from normal B6 or Fyn-/- mice at 7-wk-old age were isolated and stained for CD4 and CD8, along with CD44 or CD62L. Total CD4+ and CD8+ T cell populations were analyzed for CD44 or CD62L expression. Results are displayed as histograms of Fyn-/- mice (filled histograms) and those of normal B6 mice (open histograms). The percentage of naive CD44lowCD62Lhigh cells are indicated above the marker line (B6 mice) and below the marker line (Fyn-/- mice). One representative of five experiments is presented.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because Cbp is exclusively localized in lipid rafts and constitutively phosphorylated in resting T cells, a kinase responsible for Cbp phosphorylation should also reside and have a catalytic activity in rafts. It has been proposed that Cbp is phosphorylated by the Src family PTKs (6, 7). However, an earlier study demonstrated that Lck in the raft fraction has a low kinase activity relative to that in the nonraft fraction using Jurkat cells (13). We confirmed this finding in this study and extended it in that most Lck kinase activity is localized in the nonraft fraction not only in Jurkat cells, but also in thymocytes and peripheral T cells (Fig. 1GoB). In contrast, Fyn kinase activity is clearly detected in the raft fractions. This suggests that the catalytic activity of Fyn in rafts could be important for Cbp phosphorylation. We provided conclusive evidence in this study using Fyn-/- mice that Fyn is indeed essential for maintaining the phosphorylation level of Cbp in resting T cells. Another adaptor protein that is selectively phosphrylated by Fyn is Fyb/Slap (15). By way of phosphorylating Cbp, Fyn mediates Cbp-Csk interaction and recruits Csk to rafts. Once recruited to rafts and activated by Cbp, Csk would inhibit the kinase activity of Lck through phosphorylation of an Lck C-terminal tyrosine to keep resting T cells in a quiescent state. The Csk-Cbp complex has been shown to have a greater affinity for Src than free Csk (12). Although Fyn itself could be a target of Csk in rafts, it has been demonstrated that the kinase activity of Fyn is relatively difficult to be suppressed by Csk compared with those of other Src family PTKs (16). This may account for differential kinase activity in rafts between Lck and Fyn. Despite the predominant role of Fyn in Cbp phosphorylation, other PTKs may be involved in this process because the tyrosine phosphorylation of Cbp in T cells was not totally abrogated in Fyn-/- mice (Fig. 2Go). The residual Cbp phosphorylation in Fyn-deficient T cells could lead to reduced but significant association of Csk with Cbp (Fig. 3GoA). Further studies are necessary to determine what other kinases are contributing the phosphorylation status of Cbp.

Although Lck and Fyn are expressed at a similar level in mature T cells (17), a unique function for each PTK is not as yet well clarified. However, Fyn is known to be overexpressed in CD4-CD8- T cells from MRL/lpr mice that have an abnormal proliferative capacity and exhibit altered TCR signal transduction pathways (18). Moreover, increased Fyn kinase activity was shown to correlate with maintenance of the anergic state in T cells (19). Given that Fyn regulates an activation threshold of T cells through controlling Cbp-Csk interaction, it is possible that Csk-mediated regulation of TCR signaling could be impaired in MRL/lpr T cells or anergic T cells. Further investigation of the molecular interaction between Cbp and tyrosine kinases will provide important insights not only into physiological regulation of TCR signaling in lipid rafts but into altered T cell function in immunological diseases.


   Acknowledgments
 
We thank Dr. Masato Ogata for helpful discussions, Hisayo Ohshima for technical assistance, and Douglas R. Liddicoat for a critical review of this manuscript.


   Footnotes
 
1 This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan (12051228). Back

2 K.Y. and M.N. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Atsushi Kosugi, Faculty of Medicine, School of Allied Health Science, Osaka University, 1-7, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: kosugi{at}sahs.med.osaka-u.ac.jp Back

4 Abbreviations used in this paper: PTK, protein tyrosine kinase; Cbp, Csk-binding protein; PAG, phosphoprotein associated with glycolipid-enriched microdomains; TX, Triton X; poly EY, polymer of glutamate and tyrosine; IP, immunoprecipitate; KB, kinase buffer; CD62L, CD62 ligand. Back

Received for publication May 6, 2002. Accepted for publication July 19, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. M. Mutch, R. Sanyal, T. L. Unruh, L. Grigoriou, M. Zhu, W. Zhang, and J. P. Deans
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J. Kitaura, Y. Kawakami, M. Maeda-Yamamoto, V. Horejsi, and T. Kawakami
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M. Lovatt, A. Filby, V. Parravicini, G. Werlen, E. Palmer, and R. Zamoyska
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E. Ingley, J. R. Schneider, C. J. Payne, D. J. McCarthy, K. W. Harder, M. L. Hibbs, and S. P. Klinken
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J. Shirakawa, Y. Wang, S. Tahara-Hanaoka, S.-i. Honda, K. Shibuya, and A. Shibuya
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L. Maksumova, H. T. Le, F. Muratkhodjaev, D. Davidson, A. Veillette, and C. J. Pallen
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M.-W. Dobenecker, C. Schmedt, M. Okada, and A. Tarakhovsky
The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function
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S. Tani-ichi, K. Maruyama, N. Kondo, M. Nagafuku, K. Kabayama, J.-i. Inokuchi, Y. Shimada, Y. Ohno-Iwashita, H. Yagita, S. Kawano, et al.
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J. Shirakawa, K. Shibuya, and A. Shibuya
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S. R. Paccani, L. Patrussi, C. Ulivieri, J. L. Masferrer, M. M. D'Elios, and C. T. Baldari
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K. Sugie, M.-S. Jeon, and H. M. Grey
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L. Xing, L. T. Donlin, R. H. Miller, and K. Alexandropoulos
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V. O. Rybin, J. Guo, A. Sabri, H. Elouardighi, E. Schaefer, and S. F. Steinberg
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D. Filipp, B. L. Leung, J. Zhang, A. Veillette, and M. Julius
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G. Criado and J. Madrenas
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T. Shima, S. Nada, and M. Okada
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R. Latif, T. Ando, S. Daniel, and T. F. Davies
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S. Thomas, R. Kumar, A. Preda-Pais, S. Casares, and T.-D. Brumeanu
A Model for Antigen-Specific T-Cell Anergy: Displacement of CD4-p56lck Signalosome from the Lipid Rafts by a Soluble, Dimeric Peptide-MHC Class II Chimera
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T. Vang, H. Abrahamsen, S. Myklebust, V. Horejsi, and K. Tasken
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D. Filipp, J. Zhang, B. L. Leung, A. Shaw, S. D. Levin, A. Veillette, and M. Julius
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