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The motheaten mutation rescues B cell signaling and development in CD45-deficient mice.

Pani G, Siminovitch KA, Paige CJ - J. Exp. Med. (1997)

Bottom Line: These PTPs differ, however, in their effects on BCR function.However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45-/SHP-1-, compared to wild-type and CD45- cells.These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario, M4Y 1J3, Canada.

ABSTRACT
The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is essentially ablated in mice lacking CD45 (CD45(-)), but is enhanced in SHP-1-deficient motheaten (me) and viable motheaten (mev) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient mev (CD45-/SHP-1-) mice. Here we report that the CD45-/SHP-1-) cells undergo appropriate induction of protein kinase activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45-/SHP-1-, compared to wild-type and CD45- cells. In addition, we observed that the patterns of cell surface expression of mu, delta, and CD5, which distinguish the PTP-deficient from normal mice, are largely restored to normal levels in the double mutant animals. These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.

Show MeSH
Analysis of SHP-1 phosphoprotein binding and MAP kinase  activation in stimulated B cells from PTP-deficient mice. (A) Comparison  of protein tyrosine phosphorylation in resting and anti-Ig–treated B cells  from wild-type (CD45+/SHP-1+), CD45−CD45−/SHP-1−, and CD45+/ SHP-1− mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti–mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig–treated  B cells from wild-type (CD45+/SHP-1+), CD45−, and CD45−/SHP-1−  mice (left) and from CD45+/SHP-1− mice (right). Arrows indicate the  positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45− compared to CD45−/SHP-1−  and SHP-1− cells. Mobilities of molecular mass standards are shown on  the left. Loading of equivalent amounts of lysate protein was confirmed  by reblotting with anti–SHP-1 antibody (bottom). Data are representative  of three independent experiments on nine mice. (C) Antiphosphotyrosine  immunoblot (top) showing the tyrosine phosphorylation status of CD19  immunoprecipitates derived from biotinylated resting and anti-Ig–treated  wild-type, CD45−, and CD45−/SHP-1− cells was carried out as described in Materials and Methods. The position of CD19 is indicated by  the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after  BCR ligation in wild-type, CD45−, CD45−/SHP-1−, and CD45+/SHP-1−  cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by  anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).
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Figure 3: Analysis of SHP-1 phosphoprotein binding and MAP kinase activation in stimulated B cells from PTP-deficient mice. (A) Comparison of protein tyrosine phosphorylation in resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−CD45−/SHP-1−, and CD45+/ SHP-1− mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti–mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−, and CD45−/SHP-1− mice (left) and from CD45+/SHP-1− mice (right). Arrows indicate the positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45− compared to CD45−/SHP-1− and SHP-1− cells. Mobilities of molecular mass standards are shown on the left. Loading of equivalent amounts of lysate protein was confirmed by reblotting with anti–SHP-1 antibody (bottom). Data are representative of three independent experiments on nine mice. (C) Antiphosphotyrosine immunoblot (top) showing the tyrosine phosphorylation status of CD19 immunoprecipitates derived from biotinylated resting and anti-Ig–treated wild-type, CD45−, and CD45−/SHP-1− cells was carried out as described in Materials and Methods. The position of CD19 is indicated by the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after BCR ligation in wild-type, CD45−, CD45−/SHP-1−, and CD45+/SHP-1− cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).

Mentions: We next addressed the biochemical basis for the disparities observed in BCR-mediated activation events that distinguished CD45−/SHP-1− B cells from those found in the single PTP-deficient mice. Comparison of biochemical differences between populations of different developmental profiles should always be undertaken with caution, since the observed differences may result from a combination of direct effects due to the loss of the PTPs in question and indirect consequences that appear due to the absence of these PTPs during B cell development. Nonetheless, we compared these B cell populations by first determining their profiles of protein tyrosine phosphorylation. As is consistent with previous data linking SHP-1 to the inhibition of BCR-driven signaling cascades (13, 14) anti-Ig–induced tyrosine phosphorylation was found to be somewhat increased in the SHP-1− compared to wild-type cells (Fig. 3 A). By contrast, although CD45 has been implicated in PTK activation after antigen receptor engagement (6, 27), little difference was detected between stimulated CD45−/− and wild-type, and the CD45−/SHP-1− cells with respect to the pattern or degree of total protein tyrosine phosphorylation. The capacity of SHP-1 deficiency to rescue BCR-induced proliferative responsiveness in B cells lacking CD45 suggests a role for altered tyrosine phosphorylation of proteins that normally would be dephosphorylated by SHP-1. We therefore analyzed the tyrosine phosphorylation of proteins coprecipitated with SHP-1 from these B cell populations after BCR ligation. The results of this analysis revealed the tyrosine phosphorylation of several SHP-1–associated phosphoproteins (of ∼85–90, 115, and 120 kD) to be strikingly enhanced in both SHP-1− and CD45−/SHP-1− cells relative to wild-type B cells. By contrast, the CD45−/− cells exhibited relatively reduced tyrosine phosphorylation of SHP-1 as well as the various phosphoproteins coprecipitated with this PTP, including a 140-kD species identified by immunoblotting analysis (data not shown) as CD22, a B lineage–specific transmembrane glycoprotein implicated in the negative regulation of BCR signaling (Fig. 3 B) (16–18, 28). In view of preliminary data from our group revealing the capacity of SHP-1 to associate with CD19, a 115–120-kD cell-specific transmembrane glycoprotein that is rapidly tyrosine phosphorylated after BCR stimulation (29, 30), the possibility that CD19 is differentially phosphorylated in the context of CD45 deficiency versus CD45/SHP-1 deficiency was directly investigated. As shown by antiphosphotyrosine immunoblotting analysis of CD19 immunoprecipitates from the various PTP-deficient B cells (Fig. 3 C), CD19 phosphorylation was dramatically reduced in the CD45− cells, but increased both constitutively and after BCR ligation in the CD45−/SHP-1− cells compared to wild-type B cells. The observation of reduced CD19 phosphorylation in CD45− cells suggests a role for CD45 activity in promoting the tyrosine phosphorylation of this membrane glycoprotein. This may well be due to the effect of CD45 on the activation of the Lyn PTK, an enzyme previously shown to associate with CD19 (31). Conversely, the enhanced tyrosine phosphorylation of CD19 observed in SHP-1–deficient B cells (Fig. 3 B) suggests that SHP-1 exerts an inhibitory influence over CD19 tyrosine phosphorylation state either by direct dephosphorylation of this protein or by negative regulation of a PTK involved in CD19 phosphorylation. This interpretation is supported by the detection of a phosphoprotein that co-migrates with CD19 in SHP-1 immunoprecipitates, suggesting a capacity for SHP-1 to associate with CD19 or a CD19-bound protein. Thus, augmented phosphorylation of CD19 in the CD45−/SHP-1− cells likely reflects the loss of both SHP-1–driven CD19 dephosphorylation and CD45− dependent, PTK-mediated CD19 phosphorylation. Since CD19 has been shown to serve as a coreceptor that positively modulates signal transducing capacity of the BCR (32), these data suggest that the counterbalance of CD45 and SHP-1 effects on CD19 recruitment to the BCR signaling cascade represents one biochemical mechanism whereby these PTPs exert their coordinate regulation of BCR signaling capacity. This hypothesis, as well as the identity of the other SHP-1–associated phosphoprotein species appearing hyperphosphorylated in the CD45−/SHP-1− cells, require further investigation. Nonetheless, the suggestion that CD45 and SHP-1 coordinate effects on BCR function are realized at the level of CD19 phosphorylation is consistent with the data indicating a pivotal role for CD19 in modulating the threshold for coupling BCR stimulation to not only proliferation, but also to the development of B-1 cells (24, 33).


The motheaten mutation rescues B cell signaling and development in CD45-deficient mice.

Pani G, Siminovitch KA, Paige CJ - J. Exp. Med. (1997)

Analysis of SHP-1 phosphoprotein binding and MAP kinase  activation in stimulated B cells from PTP-deficient mice. (A) Comparison  of protein tyrosine phosphorylation in resting and anti-Ig–treated B cells  from wild-type (CD45+/SHP-1+), CD45−CD45−/SHP-1−, and CD45+/ SHP-1− mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti–mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig–treated  B cells from wild-type (CD45+/SHP-1+), CD45−, and CD45−/SHP-1−  mice (left) and from CD45+/SHP-1− mice (right). Arrows indicate the  positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45− compared to CD45−/SHP-1−  and SHP-1− cells. Mobilities of molecular mass standards are shown on  the left. Loading of equivalent amounts of lysate protein was confirmed  by reblotting with anti–SHP-1 antibody (bottom). Data are representative  of three independent experiments on nine mice. (C) Antiphosphotyrosine  immunoblot (top) showing the tyrosine phosphorylation status of CD19  immunoprecipitates derived from biotinylated resting and anti-Ig–treated  wild-type, CD45−, and CD45−/SHP-1− cells was carried out as described in Materials and Methods. The position of CD19 is indicated by  the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after  BCR ligation in wild-type, CD45−, CD45−/SHP-1−, and CD45+/SHP-1−  cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by  anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).
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Figure 3: Analysis of SHP-1 phosphoprotein binding and MAP kinase activation in stimulated B cells from PTP-deficient mice. (A) Comparison of protein tyrosine phosphorylation in resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−CD45−/SHP-1−, and CD45+/ SHP-1− mice. Loading of equivalent amounts of lysate proteins was confirmed by reblotting with anti–mb-1 antibody (bottom). (B) Antiphosphotyrosine (anti pTyr) immunoblots (top) showing the tyrosine-phosphorylated species coprecipitated with SHP-1 from resting and anti-Ig–treated B cells from wild-type (CD45+/SHP-1+), CD45−, and CD45−/SHP-1− mice (left) and from CD45+/SHP-1− mice (right). Arrows indicate the positions of SHP-1 and three associated phosphoproteins that appear differentially phosphorylated in the CD45− compared to CD45−/SHP-1− and SHP-1− cells. Mobilities of molecular mass standards are shown on the left. Loading of equivalent amounts of lysate protein was confirmed by reblotting with anti–SHP-1 antibody (bottom). Data are representative of three independent experiments on nine mice. (C) Antiphosphotyrosine immunoblot (top) showing the tyrosine phosphorylation status of CD19 immunoprecipitates derived from biotinylated resting and anti-Ig–treated wild-type, CD45−, and CD45−/SHP-1− cells was carried out as described in Materials and Methods. The position of CD19 is indicated by the arrow on the right. Loading of equivalent amounts of CD19 was confirmed by reblotting with HRP-avidin (bottom). (D) Representative example showing the levels of MAP kinase activities before and 5 min after BCR ligation in wild-type, CD45−, CD45−/SHP-1−, and CD45+/SHP-1− cells (top). Analysis of equivalent amounts of Erk-2 was confirmed by anti-Erk2 immunoblotting of equivalent aliquots of each Erk-2 immunoprecipitate (bottom).
Mentions: We next addressed the biochemical basis for the disparities observed in BCR-mediated activation events that distinguished CD45−/SHP-1− B cells from those found in the single PTP-deficient mice. Comparison of biochemical differences between populations of different developmental profiles should always be undertaken with caution, since the observed differences may result from a combination of direct effects due to the loss of the PTPs in question and indirect consequences that appear due to the absence of these PTPs during B cell development. Nonetheless, we compared these B cell populations by first determining their profiles of protein tyrosine phosphorylation. As is consistent with previous data linking SHP-1 to the inhibition of BCR-driven signaling cascades (13, 14) anti-Ig–induced tyrosine phosphorylation was found to be somewhat increased in the SHP-1− compared to wild-type cells (Fig. 3 A). By contrast, although CD45 has been implicated in PTK activation after antigen receptor engagement (6, 27), little difference was detected between stimulated CD45−/− and wild-type, and the CD45−/SHP-1− cells with respect to the pattern or degree of total protein tyrosine phosphorylation. The capacity of SHP-1 deficiency to rescue BCR-induced proliferative responsiveness in B cells lacking CD45 suggests a role for altered tyrosine phosphorylation of proteins that normally would be dephosphorylated by SHP-1. We therefore analyzed the tyrosine phosphorylation of proteins coprecipitated with SHP-1 from these B cell populations after BCR ligation. The results of this analysis revealed the tyrosine phosphorylation of several SHP-1–associated phosphoproteins (of ∼85–90, 115, and 120 kD) to be strikingly enhanced in both SHP-1− and CD45−/SHP-1− cells relative to wild-type B cells. By contrast, the CD45−/− cells exhibited relatively reduced tyrosine phosphorylation of SHP-1 as well as the various phosphoproteins coprecipitated with this PTP, including a 140-kD species identified by immunoblotting analysis (data not shown) as CD22, a B lineage–specific transmembrane glycoprotein implicated in the negative regulation of BCR signaling (Fig. 3 B) (16–18, 28). In view of preliminary data from our group revealing the capacity of SHP-1 to associate with CD19, a 115–120-kD cell-specific transmembrane glycoprotein that is rapidly tyrosine phosphorylated after BCR stimulation (29, 30), the possibility that CD19 is differentially phosphorylated in the context of CD45 deficiency versus CD45/SHP-1 deficiency was directly investigated. As shown by antiphosphotyrosine immunoblotting analysis of CD19 immunoprecipitates from the various PTP-deficient B cells (Fig. 3 C), CD19 phosphorylation was dramatically reduced in the CD45− cells, but increased both constitutively and after BCR ligation in the CD45−/SHP-1− cells compared to wild-type B cells. The observation of reduced CD19 phosphorylation in CD45− cells suggests a role for CD45 activity in promoting the tyrosine phosphorylation of this membrane glycoprotein. This may well be due to the effect of CD45 on the activation of the Lyn PTK, an enzyme previously shown to associate with CD19 (31). Conversely, the enhanced tyrosine phosphorylation of CD19 observed in SHP-1–deficient B cells (Fig. 3 B) suggests that SHP-1 exerts an inhibitory influence over CD19 tyrosine phosphorylation state either by direct dephosphorylation of this protein or by negative regulation of a PTK involved in CD19 phosphorylation. This interpretation is supported by the detection of a phosphoprotein that co-migrates with CD19 in SHP-1 immunoprecipitates, suggesting a capacity for SHP-1 to associate with CD19 or a CD19-bound protein. Thus, augmented phosphorylation of CD19 in the CD45−/SHP-1− cells likely reflects the loss of both SHP-1–driven CD19 dephosphorylation and CD45− dependent, PTK-mediated CD19 phosphorylation. Since CD19 has been shown to serve as a coreceptor that positively modulates signal transducing capacity of the BCR (32), these data suggest that the counterbalance of CD45 and SHP-1 effects on CD19 recruitment to the BCR signaling cascade represents one biochemical mechanism whereby these PTPs exert their coordinate regulation of BCR signaling capacity. This hypothesis, as well as the identity of the other SHP-1–associated phosphoprotein species appearing hyperphosphorylated in the CD45−/SHP-1− cells, require further investigation. Nonetheless, the suggestion that CD45 and SHP-1 coordinate effects on BCR function are realized at the level of CD19 phosphorylation is consistent with the data indicating a pivotal role for CD19 in modulating the threshold for coupling BCR stimulation to not only proliferation, but also to the development of B-1 cells (24, 33).

Bottom Line: These PTPs differ, however, in their effects on BCR function.However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45-/SHP-1-, compared to wild-type and CD45- cells.These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Toronto, Toronto, Ontario, M4Y 1J3, Canada.

ABSTRACT
The cytosolic SHP-1 and transmembrane CD45 protein tyrosine phosphatases (PTP) play critical roles in regulating signal transduction via the B cell antigen receptor (BCR). These PTPs differ, however, in their effects on BCR function. For example, BCR-mediated mitogenesis is essentially ablated in mice lacking CD45 (CD45(-)), but is enhanced in SHP-1-deficient motheaten (me) and viable motheaten (mev) mice. To determine whether these PTPs act independently or coordinately in modulating the physiologic outcome of BCR engagement, we assessed B cell development and signaling in CD45-deficient mev (CD45-/SHP-1-) mice. Here we report that the CD45-/SHP-1-) cells undergo appropriate induction of protein kinase activity, mitogen-activated protein kinase activation, and proliferative responses after BCR aggregation. However, BCR-elicited increases in the tyrosine phosphorylation of several SHP-1-associated phosphoproteins, including CD19, were substantially enhanced in CD45-/SHP-1-, compared to wild-type and CD45- cells. In addition, we observed that the patterns of cell surface expression of mu, delta, and CD5, which distinguish the PTP-deficient from normal mice, are largely restored to normal levels in the double mutant animals. These findings indicate a critical role for the balance of SHP-1 and CD45 activities in determining the outcome of BCR stimulation and suggest that these PTPs act in a coordinate fashion to couple antigen receptor engagement to B cell activation and maturation.

Show MeSH