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Involvement of LAT, Gads, and Grb2 in compartmentation of SLP-76 to the plasma membrane.

Ishiai M, Kurosaki M, Inabe K, Chan AC, Sugamura K, Kurosaki T - J. Exp. Med. (2000)

Bottom Line: Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells.This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking.In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan.

ABSTRACT
B cell linker protein (BLNK) and Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76) are adaptor proteins required for B cell receptor (BCR) and T cell receptor function, respectively. Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells. This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking. Supporting this idea, the BCR function was restored when a membrane-associated SLP-76 chimera was enforcedly localized to GEMs. Moreover, we demonstrate that addition of both linker for activation of T cells (LAT) and Grb2-related adaptor downstream of Shc (Gads) to SLP-76 allow SLP-76 to be recruited into GEMs, whereby the BCR function is reconstituted. The Gads function was able to be replaced by overexpression of Grb2. In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.

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Western blot analysis of GEM fractions. After stimulation by M4 for 3 min, GEMs were prepared from E42-11 or E48-1 cells (4 × 108) by sucrose density gradient centrifugation. 40 μl of the individual fractions, except for fractions 11 and 12 (2 μl), were analyzed by Western blotting using anti-FLAG mAb, anti-Lyn Ab, or antitubulin mAb.
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Figure 2: Western blot analysis of GEM fractions. After stimulation by M4 for 3 min, GEMs were prepared from E42-11 or E48-1 cells (4 × 108) by sucrose density gradient centrifugation. 40 μl of the individual fractions, except for fractions 11 and 12 (2 μl), were analyzed by Western blotting using anti-FLAG mAb, anti-Lyn Ab, or antitubulin mAb.

Mentions: It has been recently shown that BCR and the associated Igα rapidly translocate to GEMs upon receptor cross-linking, which is required for efficient signal transduction by the BCR 458. We therefore compared translocation of BLNK and SLP-76 into GEMs upon BCR cross-linking. After receptor stimulation, DT40 B cells were solubilized with 0.5% Triton X-100, and lysates were subjected to supercentrifiguration over a sucrose density gradient. As reported previously 45, Lyn was enriched in the fraction 4, and tubulin was found to be completely excluded from the GEMs (Fig. 2). Although the stoichiometry of GEM-associated BLNK was substantially enhanced after BCR ligation, the significant translocation of SLP-76 to the GEMs could not be detected (Fig. 2). Taken together, these two lines of findings suggest that the failure of SLP-76 to evoke PLC-γ2 activation in B cells is due to its lower phosphorylation and/or its inability to be recruited into GEMs.


Involvement of LAT, Gads, and Grb2 in compartmentation of SLP-76 to the plasma membrane.

Ishiai M, Kurosaki M, Inabe K, Chan AC, Sugamura K, Kurosaki T - J. Exp. Med. (2000)

Western blot analysis of GEM fractions. After stimulation by M4 for 3 min, GEMs were prepared from E42-11 or E48-1 cells (4 × 108) by sucrose density gradient centrifugation. 40 μl of the individual fractions, except for fractions 11 and 12 (2 μl), were analyzed by Western blotting using anti-FLAG mAb, anti-Lyn Ab, or antitubulin mAb.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193288&req=5

Figure 2: Western blot analysis of GEM fractions. After stimulation by M4 for 3 min, GEMs were prepared from E42-11 or E48-1 cells (4 × 108) by sucrose density gradient centrifugation. 40 μl of the individual fractions, except for fractions 11 and 12 (2 μl), were analyzed by Western blotting using anti-FLAG mAb, anti-Lyn Ab, or antitubulin mAb.
Mentions: It has been recently shown that BCR and the associated Igα rapidly translocate to GEMs upon receptor cross-linking, which is required for efficient signal transduction by the BCR 458. We therefore compared translocation of BLNK and SLP-76 into GEMs upon BCR cross-linking. After receptor stimulation, DT40 B cells were solubilized with 0.5% Triton X-100, and lysates were subjected to supercentrifiguration over a sucrose density gradient. As reported previously 45, Lyn was enriched in the fraction 4, and tubulin was found to be completely excluded from the GEMs (Fig. 2). Although the stoichiometry of GEM-associated BLNK was substantially enhanced after BCR ligation, the significant translocation of SLP-76 to the GEMs could not be detected (Fig. 2). Taken together, these two lines of findings suggest that the failure of SLP-76 to evoke PLC-γ2 activation in B cells is due to its lower phosphorylation and/or its inability to be recruited into GEMs.

Bottom Line: Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells.This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking.In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan.

ABSTRACT
B cell linker protein (BLNK) and Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76) are adaptor proteins required for B cell receptor (BCR) and T cell receptor function, respectively. Here, we show that expression of SLP-76 cannot reconstitute BCR function in Zap-70(+)BLNK(-) B cells. This could be attributable to inability of SLP-76 to be recruited into glycolipid-enriched microdomains (GEMs) after antigen receptor cross-linking. Supporting this idea, the BCR function was restored when a membrane-associated SLP-76 chimera was enforcedly localized to GEMs. Moreover, we demonstrate that addition of both linker for activation of T cells (LAT) and Grb2-related adaptor downstream of Shc (Gads) to SLP-76 allow SLP-76 to be recruited into GEMs, whereby the BCR function is reconstituted. The Gads function was able to be replaced by overexpression of Grb2. In contrast to SLP-76, BLNK did not require Grb2 families for its recruitment to GEMs. Hence, these data suggest a functional overlap between BLNK and SLP-76, while emphasizing the difference in requirement for additional adaptor molecules in their targeting to GEMs.

Show MeSH
Related in: MedlinePlus