MHC-II MAb induces time and dosage-dependent death of K46 B lymphoma cells.
We studied MHC-II-induced cell death in a murine B lymphoma line, K46 (17
). Cross-linking of MHC-II by MAbs in K46 cells has been shown to induce tyrosine phosphorylation, calcium flux, and phosphatidylinositol 3-kinase activation (3
). As shown in Fig. , MHC-II cross-linking by biotinylated I-Ab,d,q
-reactive MAb M5/114 (rat IgG2b) and avidin induced time and dosage-dependent K46 B lymphoma exposure of phosphatidylserine as indicated by annexin V staining. The anti-Fc receptor MAb 2.4G2 (rat IgG2b) was used as an isotype control. This MHC-II MAb response, which suggests a commitment to cell death, was observed as early as 1 h and peaked at 5 h (Fig. ). The cell death response was also measured by dual staining with PI and DiOC6(3)/PI (Fig. ). PI staining reflects cell permeability, and loss of DiOC6(3) staining reflects mitochondrial membrane depolarization. The control 2.4G2 MAb did not induce cell death. Our findings demonstrate that MHC-II molecules transduce apoptotic cell death signals in K46 cells and this response involves depolarization of mitochondrial membranes.
FIG. 1. MHC class II MAb induces time and dosage-dependent apoptosis of K46 B lymphoma cells. (A) K46 cells were treated with 15 μg/ml biotinylated anti-MHC class II (M5/114; rat IgG2b) MAb or its isotype control, biotinylated anti-mouse FcγRIIB (more ...) PP2 inhibits MHC-II MAb-induced tyrosine phosphorylation and calcium mobilization but not the death response.
Cross-linking of MHC-II by MAbs in K46 cells is known to induce protein tyrosine phosphorylation and calcium flux (3
). To explore whether these signaling events are involved in the death response, we assessed the effect of the Src kinase inhibitor PP2 on induction of cell death. As expected, MHC-II MAb-induced tyrosine phosphorylation and calcium responses were blocked by PP2 treatment (Fig. ). However, MHC-II MAb-induced cell death remained largely intact after treatment with inhibitor (Fig. ). This finding is consistent with results in previous studies, which showed that tyrosine phosphorylation and calcium flux are not required for the MHC-II-mediated death response of human B cells (14
FIG. 2. MHC-II signaling of cell death does not require activation of Src family tyrosine kinases. (A) K46 cells were treated with 25 μM PP2 or dimethylsulfoxide (DMSO) for 30 min at 37°C and then stimulated with biotin-MHC-II (M5.114; 20 μg/ml) (more ...) MHC-II MAb-induced ERK activation but not AKT and p38 activation is required for the death response.
MHC-II aggregation can lead to activation of AKT and mitogen-activated protein kinase kinases ERK, p38, and JNK in monocytes and human B cells (1
). We investigated whether these signaling events are activated upon MHC-II MAb stimulation of K46 cells. Indeed, cross-linking of MHC-II by biotinylated M5/114 and avidin induced strong and sustained ERK and p38 activation in K46 cells (Fig. ). AKT was weakly activated by MHC-II cross-linking, as measured using phospho-AKT Ab blotting (Fig. ). There was weak basal JNK activation measured by phosphor-JNK Ab. However, MHC-II activation did not increase JNK phosphorylation (Fig. ). Thus, MHC-II cross-linking activates AKT, ERK, and p38 but not JNK in K46 cells.
FIG. 3. ERK function is required for MHC-II-mediated cell death. (A to C) K46 cells were stimulated with biotin-MHC-II (M5/114; 20 μg/ml) plus avidin (20 μg/ml) for the indicated times and detergent lysates prepared. Transfers of SDS-PAGE-fractionated (more ...)
We then studied requirements for these signaling events in the cell death induced by MHC-II MAb. Treatment of K46 cells with LY294002 inhibited MHC-II-mediated AKT activation but had no effect on the death response (Fig. ). MHC-II-mediated p38 activation was completely inhibited by SB203580, but the inhibitor also had no effect on the death response (Fig. ). Interestingly, at 50 μM, the ERK-specific inhibitor PD98059 inhibited MHC-II-mediated cell death by ~50% (Fig. ). However, this dose did not completely inhibit MHC-II-induced ERK activation, explaining the residual cell survival and implicating ERK in the cell death response (Fig. ).
In conclusion, although cross-linking MHC-II with MAb leads to activation of AKT, ERK, and p38, only ERK activation is required for the death response of these cells.
Identification of a novel MHC-II-associated membrane protein using nano-LC-MS/MS.
The signaling circuitry by which MHC-II aggregation activates ERK is unknown. MHC-II α and β chains contain cytoplasmic tails of only 12 and 18 amino acids, respectively, and deletion of these tails does not affect the death response (16
). Thus, we postulated that MHC-II must transmit death signals through an associated cell surface protein(s). The B-cell-specific proteins CD19, CD20, and CD79a/b have been shown to be physically and functionally associated with MHC-II and therefore were candidates (18
). However, unlike the MHC-II death response, CD19, CD20, and CD79a/b expression is B cell specific, suggesting that these molecules are not likely to be involved in this response. MHC-II is also known to associate with tetraspanins, including CD9 and CD37 (19
). However, these molecules have very short cytoplasmic tails (8 to ~14 aa) that lack defined signaling motifs. Therefore, we hypothesized that a novel MHC-II-associated protein(s) might function in transduction of signals that mediate the death response. To explore this possibility, we undertook proteomic analysis of MHC-II-associated proteins. We prepared lysates of K46 cells using a mild detergent (CHAPS) that preserves weak protein-protein interactions and then immunoprecipitated MHC-II and associated proteins using anti-MHC-II MAb beads. We eluted proteins from the beads and identified them using nano-LC-MS/MS. Forty-one proteins were recovered from the MHC-II immunoprecipitates based on the detection of at least two unique peptides from each (see Table S1 in the supplemental material). MHC-II α and β chains were identified by 6 and 14 peptides, respectively, confirming the effectiveness of the affinity purification (see Fig. S1a in the supplemental material).
We were particularly interested in MHC-II-associated proteins containing a transmembrane domain(s) and cytoplasmic signaling motifs. A hypothetical protein, RIKEN cDNA 2610307O08, was implicated by three unique peptides predicted by its DNA sequence (see Fig S1a in the supplemental material). This hypothetical protein is predicted to contain four transmembrane domains by the SOSHI (http://bp.nuap.nagoya-u.ac.jp/sosui/
), TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/
), and TMpred (http://www.ch.embnet.org/software/TMPRED_form.html
) software programs, along with multiple signaling motifs predicted by the ELM (http://elm.eu.org/
) and Scansite (http://scansite.mit.edu
) software programs, including an immunoreceptor tyrosine-based inhibitory motif (ITIM), SVY244
EIL) (Fig. ). We designated it MPYS based on its N-terminal methionine-proline-tyrosine-serine amino acid sequence.
FIG. 4. Identification of a novel MHC-II-associated membrane protein. MHC-II-associated proteins were isolated by coimmunoprecipitation from K46 cell CHAPS detergent lysates and analyzed using nano-LC-MS/MS. One candidate, designated MPYS, was identified based (more ...)
We cloned the mpys
gene from a cDNA library produced from the murine K46 B lymphoma cell line (17
). The gene encoded a 378-aa protein with a predicted mass of 42 kDa (Fig. ). The sequence does not show significant homology to known or predicted proteins, suggesting MPYS belongs to a novel, single-member class of proteins. Human MPYS is ~80% homologous with mouse MPYS, and no invertebrate homologues of mpys
were found in the database (Fig. ).
To begin to explore protein expression and function, we raised a polyclonal Ab against the C-terminal 101 aa of murine MPYS and used it to immunoprecipitate endogenous MPYS from K46 cell lysates. We then analyzed eluates by nano-LC-MS/MS. Ab reactivity with MPYS was confirmed by detection in eluates of 15 unique peptides covering more than 50% of the total amino acid sequence (see Fig. S1b in the supplemental material), including peptides from the N and C termini of the protein (see Fig. S1b in the supplemental material). Consistent with MPYS association, two and four peptides derived from MHC-II α and β chains, respectively, were found in the immunoprecipitate (see Fig. S1b in the supplemental material). This association was confirmed by immunoblotting (Fig. ).
To evaluate the cell surface expression of MPYS, we expressed mpys-HA in K46 cells. These cells are designated KHA. Anti-HA immunoprecipitation (IP) of lysates of surface-biotinylated K46 cells, followed by SDS-PAGE, transfer, and avidin blotting, revealed that MPYS is biotinylated (Fig. ) and therefore is on the cell surface.
To confirm the predicted topology of the MPYS protein, we made two Flag-tagged mpys constructs. One has a Flag tag inserted in the predicted large extracellular loop (LEL-Flag). The other has a Flag tag in the predicted small extracellular loop (SEL-Flag). We expressed these two constructs in K46 cells and stained the cells with anti-Flag Ab. As shown in Fig. , the Flag-tagged MPYS proteins were detected on the cell surface. On the contrary, our own polyclonal Ab, which is against the predicted cytoplasmic tail of MPYS, did not stain intact cells (data not shown). These data suggest that the predicted four-transmembrane topology is likely correct (Fig. ).
To investigate MPYS distribution in cells, we made a MPYS-GFP fusion construct and expressed it in K46 cells. Confocal microscopy showed that while some MPYS was found on the cell surface, a large proportion was actually localized to mitochondria (Fig. ).
The transmembrane region of MPYS contains four charged residues and two cysteines. Such residues often mediate inter- or intraprotein interactions. To determine if MPYS can form protein complexes, we used the membrane-permeative chemical cross-linker dithiobis(succinimidyl)propionate to form covalent bonds between neighboring proteins and performed SDS-PAGE analysis on K46 whole-cell lysates on a nonreducing SDS-PAGE gel. In addition to the monomer, MPYS immunoblotting revealed an ~80-kDa band, a band double the size of the MPYS monomer (Fig. ). This suggested that most MPYS exists as a dimer within cells. The blot was stripped and reblotted with MPYS Abs in the presence of a blocking peptide (the 101 aa used to generate the MPYS Ab). As show in Fig. , no protein bands were recognized, indicating that MPYS Ab blotting is specific. Sequential blotting for CD19 provided an additional control for equivalent loading (Fig. ).
To assess tissue distribution of MPYS, we probed SDS-PAGE fractionated and transferred lysates of various B and T cells (Fig. ). Anti-MPYS reacted with a predominant 40-kDa species in spleen and thymus tissue (Fig. ). Splenocytes have higher MPYS expression than thymocytes, which is consistent with higher level-expression in B cells (Fig. ). MPYS was also present in dendritic cells (Fig. ).
To assess potential changes in MPYS expression during B-cell development and differentiation, whole-cell lysates from B-lineage tumors were probed with MPYS Ab. MPYS Ab recognized a 40-kDa band that was enhanced in K46 cells expressing the HA-tagged mpys gene (Fig. ). MPYS was highly expressed in cells representing mature stages of B cells (Bal17 line) but weakly expressed in pre-B cells (70Z/3 line), immature B cells (WEHI 231 line), and memory B-cell stages (A20 line). It was not detected in plasma cells (J558L line) (Fig. ). Thus, it appears to be expressed throughout the B lineage prior to the plasma cell stage but occurs at highest levels in mature B cells.
MPYS possesses inhibitory signaling function.
The cytoplasmic tail of MPYS contains ITIMs, motifs known to recruit the inhibitory signaling effectors SHP-1 and SHIP (11
). To assess the ability of MPYS to serve an inhibitory function, K46 cells were stimulated with anti-MHC-II MAb for 2 min before cells were lysed and MPYS was immunoprecipitated, fractionated by SDS-PAGE, and subjected to immunoblotting analysis. Antiphosphotyrosine blotting revealed that MPYS is tyrosine phosphorylated upon MHC-II cross-linking (Fig. ). Reprobing the blot with anti-SHP-1 and SHIP Abs showed that phosphorylated MPYS bound SHP-1 and SHIP (Fig. ). Notably, a 32-kDa unknown tyrosine-phosphorylated protein was also associated with MPYS. Thus, MPYS engages negative signaling effectors when tyrosine is phosphorylated, consistent with its ITIM.
FIG. 5. MPYS negatively regulates MHC-II signaling. (A) K46 cells were stimulated using biotinylated anti-MHC-II (M5/114, 20 μg/ml) and avidin (40 μg/ml) for 2 min. Cells were lysed in CHAPS buffer. MPYS was immunoprecipitated using MPYS polyclonal (more ...)
Studies of various inhibitory receptors suggest that SHP-1 and SHIP recruitment results in inhibition of calcium mobilization (11
). To assess whether MPYS mediates this function, we overexpressed MPYS in K46 cells (Fig. ). MPYS overexpression led to dramatically reduced MHC-II-mediated calcium mobilization (Fig. ). These data suggest that MPYS act in feedback regulation of some MHC-II signals, i.e., those that lead to calcium mobilization.
We also found that cells overexpressing MPYS tend to be lost from populations during culture, suggesting MPYS may have a negative effect on cell growth. To further explore this possibility, we expressed an MPYS-GFP fusion construct in A20 cells. Sorted MPYS-GFP-positive A20 B lymphoma cells lost MPYS-GFP expression within 10 days (Fig. ). In contrast, expression of GFP alone in A20 was maintained indefinitely (data not shown). These data indicate that MPYS functions as a negative regulator of cell growth/viability.
Knockdown of MPYS expression in K46 cells inhibits MHC class II aggregation-induced cell death and ERK activation.
To further study the role of MPYS in MHC-II signaling, an shRNA targeting exon 5 (sh5) or exon 7 (sh7) of the mpys gene was prepared and used to knock down MPYS expression in K46 cells. Cells expressing sh5 RNA displayed a >90% reduction in MPYS expression, while in cells expressing sh7-RNA MPYS, expression decreased by >80% (Fig. ). Surface expression of IgM, MHC-II, CD19, CD45, CD80, and CD86 was not altered by shRNA expression (data not shown). Contrary to the case with MPYS overexpression (Fig. ), MPYS knockdown increased the growth rate of K46 cells (Fig. ).
FIG. 6. Knockdown of MPYS expression in K46 cells inhibits MHC class II-mediated cell death and ERK activation. (A) K46 cells expressing the control shRNA targeting the luciferase gene (luc) or exon 5 of the mpys gene (sh5) or exon 7 of the mpys gene (sh7) were (more ...)
We next examined the role of MPYS in the death response by assessing anti-MHC-II induction of death in K46 cells expressing MPYS knockdown constructs. K46 cells expressing a control luciferase shRNA (luc) or MPYS sh5 knockdown shRNA (sh5) were stimulated with biotinylated MAb M5/114 and avidin, and cell death was measured by annexin V/PI dual staining (Fig. ). Biotinylated MAb 2.4G2 was used as an isotype control. MHC-II MAb-induced cell death was reduced significantly in K46 cells expressing the sh5 MPYS construct (where MPYS expression is diminished by >90%) (Fig. ). An effect of MPYS knockdown was noted at all doses of anti-MHC-II MAb used (Fig. ). Similar results were observed in K46 cells expressing the sh7 MPYS knockdown construct (data not shown). The fact that two shRNAs, targeting different regions of mpys, caused similar outcomes argues strongly that this is not an off-target effect. Similar results were observed when other anti-MHC-II Abs were used for stimulation (data not shown). We conclude that MPYS expression is essential for anti-MHC-II MAb induction of B-cell death.
To extend earlier findings that MHC-II-mediated activation of ERK is required for the death response, we investigated the effect of MPYS knockdown on ERK activation. We probed fractionated and transferred lysates of luc or sh5 shRNA-expressing K46 cells using phospho-ERK Ab. MPYS knockdown inhibited MHC-II MAb-induced ERK activation (Fig. ). These findings indicate that MHC class II signaling of cell death is dependent on MPYS-linked ERK activation.
MHC-II-mediated, MPYS-dependent activation of ERK is Src family kinase independent.
If, as shown in Fig. , anti-MHC class II-induced death is not dependent on Src family kinase-mediated tyrosine phosphorylation but is ERK dependent, one would predict that ERK activation by class II signals should not be inhibited by PP2. As shown in Fig. , this is the case. Taken together, these data indicate that the death response is mediated by Src family kinase-independent activation of MPYS and downstream ERK. Further, PP2-sensitive anti-class II-activated signaling events, including calcium mobilization and MPYS tyrosine phosphorylation and association with SHIP-1 and SHP-1, are not required for the death response (Fig. ; also data not shown).
Finally, we addressed whether the MPYS dependence of ERK activation reflects a requirement that MPYS interact directly with ERK. Cells were activated with MHC-II MAb before being lysed and subjected to MPYS immunoprecipitation. Immunoprecipitates were analyzed by SDS-PAGE and ERK immunoblotting. Under conditions in which we observed SHP-1 recruitment to MPYS, we did not detect recruitment of ERK (Fig. ).
MHC-II-independent aggregation of MPYS leads to cell death signaling.
To test the ability of MPYS to transduce death signals when aggregated independently, we stimulated K46 cells expressing Flag-tagged MPYS (LEL-MPYS) with anti-Flag and measured death by PI staining. As shown in Fig. , anti-Flag treatment led to a 10-fold increase in PI staining of cells within 15 h of stimulation. This response, while clearly significant and Ab dose dependent, was somewhat less than that induced by anti-MHC-II. We conclude that MPYS may function in isolation as a transducer of death signals and thus could mediate death signaling in MHC-II-negative cells, such as T cells.
FIG. 7. Aggregation of surface MPYS induces K46 cell death. (A) K46 cells expressing the Flag-tagged-MPYS (LEL-MPYS) were stimulated for 15 h with different doses of 2.4G2 anti-FcR MAb (FcR), anti-Flag MAb (MPYS), or D3.137 anti-MHC-II MAb (MHC II). Cell death (more ...)