The HS1 protein is highly tyrosine-phosphorylated upon BCR cross-linking (7
), probably by Lyn and/or Syk. Because tyrosine phosphorylation of various cellular proteins was greatly enhanced in fibroblasts transfected with both Lyn and Syk expression plasmids as compared with those transfected with either the Lyn or Syk plasmid alone (6
), we examined whether Lyn and Syk can synergistically phosphorylate HS1 in the cells. The results showed that HS1 was highly tyrosine-phosphorylated only when both Lyn and Syk were coexpressed (Fig. ). This is consistent with our previous observation that another Src family member, Fyn, cooperates with ZAP-70, an analogue of Syk, in phosphorylating HS1 in T cells (12
). It has been shown that Lyn activates Syk when they are coexpressed in fibroblasts (6
) and that BCR cross-linking induces little activation of Syk in the splenic B cells (Nishizumi, H., unpublished data) and mast cells (16
) of lyn−/−
mice. Accordingly, lyn−/−
splenocytes failed to induce tyrosine phosphorylation of HS1 upon BCR cross-linking (4
). In contrast, Lyn is not detectably activated by Syk (6
). Therefore, our results suggest that Syk activated by Lyn phosphorylates HS1 directly in BCRmediated signaling.
Figure 1 Cooperation between Lyn and Syk in HS1 phosphorylation. CV-1 cells (1.5 × 106) were transfected with the expression plasmids (5 μg), pMELyn (+), pME-Syk (+), pMEHS1 (+), or with H2O in place of DNA solution (−), (more ...)
Of the 17 tyrosine residues in human HS1, Tyr-378 and Tyr-397 are preceded by acidic residues (ENDY378
), which are characteristic of many tyrosine phosphorylation sites (17
). In fact, the EGDY397
EEV sequence of HS1 is the best known substrate for Syk kinase (19
). Because tyrosine-phosphorylated HS1 interacts with the SH2 domains of Src family kinases (7
), and because a phosphorylated pYED/E sequence shows the highest affinity for these domains (20
), we predicted that the relevant phosphorylation sites on HS1 would be followed by two acidic residues. Of all the tyrosine residues in HS1, only Tyr-378 and Tyr-397 match the consensus (Y378
ED and Y397
EE) (Fig. A
). These amino acids are also conserved in mouse HS1 (21
). Thus, Tyr-378 and Tyr-397 of human HS1 are likely phosphorylated by the BCR-associated kinases. Indeed, when coexpressed with Lyn and Syk in CV-1 cells, a deletion mutant HS1-ΔYY, lacking 23 amino acids from Tyr-378 to Val-400, was tyrosine phosphorylated at a greatly reduced level compared with wild-type HS1 (Fig. B
Figure 2 Requirement of Tyr-378 and Tyr-397 in tyrosine phosphorylation of HS1 upon BCR stimulation. (A) Schematic structure of the human HS1 protein. The 23 amino acids sequence of HS1, Tyr-378 to Val-400, deleted to generate HS1-ΔYY, is shown on the (more ...)
To verify BCR-mediated phosphorylation on Tyr-378 and Tyr-397 of HS1, we generated three HS1 mutants, HS1-FY, HS1-YF, and HS1-FF, in which Tyr-378, Tyr397, and both have been substituted by phenylalanine, respectively. These mutants and wild-type HS1 were expressed in WEHI-231 cells by retroviral infection. By probing the anti-human HS1 immunoprecipitates with the antiphosphotyrosine antibody, we showed that HS1-FF was not detectably tyrosine-phosphorylated upon BCR crosslinking, whereas wild-type HS1 was highly and rapidly tyrosine-phosphorylated (Fig. C). Both HS1-FY and HS1-YF were phosphorylated at a very low level. Thus, Tyr-378 and Tyr-397 are important for tyrosine phosphorylation of HS1 upon BCR stimulation.
WEHI-231–derived M1 cells are resistant to mIgMinduced apoptosis, unlike their parental cells. This resistibility is due to very low expression of HS1 in the cells (9
). M1 cells are rendered sensitive to BCR-mediated apoptosis by the exogenous expression of wild-type HS1 (9
). Furthermore, peritoneal B cells from HS1−/−
mice do not undergo apoptosis upon BCR cross-linking (8
). These data indicate that HS1 is a critical molecule for BCR-mediated apoptosis. However, unlike wild-type HS1, the exogenously introduced HS1-FF protein failed to restore the sensitivity of M1 cells to BCR-mediated apoptosis (Fig. A
). This was not due to differences in the expression levels of these proteins. In addition, the HS1-FF protein in M1 cells, as in WEHI-231 cells, was not tyrosine-phosphorylated by BCR cross-linking (Fig. B
). Therefore, tyrosine phosphorylation of HS1 is essential for BCR-mediated apoptosis.
Figure 3 HS1 tyrosine phosphorylation is required for BCR-mediated apoptosis. (A) Restoration of BCR-mediated apoptosis by exogenous HS1 but not by HS1-FF in M1 cells. WEHI-231 cells (1 and 2), M1 cells (3 and 4), M1-derived cells expressing wild-type HS1 (more ...)
Despite the presence of a putative nuclear localization signal, HS1 localizes mainly in the cytoplasm of resting B cells (7
). Consistently, HS1 expressed in COS7 cells was present in the cytoplasm. However, HS1 coexpressed together with Lyn and Syk was mostly in the nucleus. In contrast, HS1-FF remained in the cytoplasm in the presence of Lyn and Syk (Fig. A
). Thus, tyrosine phosphorylation of HS1 appears to be required for its own translocation from the cytoplasm to the nucleus. These data suggest that BCR cross-linking causes a significant fraction of tyrosine-phosphorylated HS1 to localize to the nucleus. Consistently, subcellular localization experiments showed that the amount of HS1 in the nucleus is increased after BCR cross-linking (Fig. B
). Similarly, the amount of wild-type human HS1 but not HS1-FF mutant expressed in M1 cells was increased in the nuclei upon BCR stimulation (Nishizumi, H., unpublished data). Tyrosine phosphorylation on HS1 may trigger its conformational alteration that allows its nuclear translocation and thereby signaling to its downstream targets.
Figure 4 Subcellular localization of HS1. (A) Nuclear localization of HS1 coexpressed with Lyn and Syk. COS7 cells (1.5 × 106) were transfected with the expression plasmids (5 μg), pME-Lyn, pME-Syk, and either pME-HS1 (b), or pME-HS1-FF (c (more ...)
Because de novo protein synthesis is required for BCRmediated apoptosis of WEHI-231 cells (22
), the transcriptional and/or translational regulation of an as yet unidentified gene(s) is involved in the process. HS1 possesses motifs characteristic of transcription factors (11
). Therefore, it may regulate gene expression as a transcription factor following translocation into the nucleus, as is proposed for the STAT family of proteins. Alternatively, HS1 may interact with the other transcription factors or may transport a protein(s) critical for apoptosis into the nucleus. Molecules that may interact with the other motifs, such as the SH3 domain, of HS1 have yet to be determined.
Basing on the present data we propose a model that, upon BCR cross-linking, Syk becomes fully activated by Lyn and phosphorylates HS1 on Tyr-378 and Tyr-397. Because the Lyn SH2 domain binds tyrosine-phosphorylated HS1 (7
), phosphorylated Y378
ED and Y397
EE could be the binding sites. This interaction would then allow Lyn to phosphorylate HS1 on other tyrosine residues. The model is consistent with the sequential phosphorylation model in which the primary kinase phosphorylates a residue that is directly recognized by the secondary phosphate-directed kinase. The secondary kinase then phosphorylates another residue nearby (23
). A similar mechanism is proposed for tyrosine phosphorylation of p130CAS
by Abl (24
). Accordingly, not only wild-type HS1 but also HS1-YF and HS1FY would be phosphorylated upon BCR stimulation on Tyr-378 and Tyr-397, respectively, and thereby would interact with Lyn, allowing their further phosphorylation. However, HS1-YF and HS1-FY were much less tyrosine phoshorylated than wild-type HS1 (Fig. C
), as though no secondary kinases were available. It should be noted that Syk can interact with unphosphorylated HS1 and that the interaction terminates once the HS1 protein becomes tyrosine-phosphorylated at appropriate sites (Fukuda, T., unpublished data). This allows us to speculate that Lyn interacts with HS1 only when the two residues, Tyr-378 and Tyr-397, become phosphorylated and HS1 is dissociated from Syk. It is likely that HS1-YF and HS1-FY are still associated with Syk even after BCR stimulation, which prevents Lyn from interacting with HS1. Once the two tyrosine residues are both phosphorylated, processive phosphorylation of HS1 by Lyn and the other Src family kinases would take place, producing hyperphosphorylated form of HS1. Finally, it is this hyperphosphorylated form of HS1 that translocates to the nucleus and activates B cell apoptosis.