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Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling.

Qian D, Lev S, van Oers NS, Dikic I, Schlessinger J, Weiss A - J. Exp. Med. (1997)

Bottom Line: Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation.The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro.Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-zeta chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.

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Fyn, but not Lck, is  required for TCR-stimulated  Pyk2 tyrosine phosphorylation.  (A) Pyk2 phosphorylation in Jurkat T cells. Left, Jurkat cells were  stimulated with anti-TCR  (mAb C305) for the indicated  period of times. Right, Jurkat  cells were stimulated with antiTCR or ionomycin (IONO) for  2 min. Cells were lysed, Pyk2  was immunoprecipitated with  anti-Pyk2 (antibody no. 3), and  analyzed by immunoblotting using anti–P-Tyr mAb RC20. The  level of Pyk2 in each sample was  verified either by immunoblotting an aliquot of anti-Pyk2 immunoprecipitates with antiPyk2 (antibody no. 3) (left) or by  stripping and reprobing the same  blot with anti-Pyk2 (right). (B)  Thymocytes isolated from wildtype (Wt), lck−/−, or fyn−/− mice  were either left unstimulated or  stimulated with the anti-CD3  mAb for 3 min. Cells were lysed,  Pyk2 was immunoprecipitated  with anti-Pyk2 (antibody no.  600), and analyzed by immunoblotting using anti-P-Tyr mAb  4G10. The same blot was  stripped and reprobed with antiPyk2 (antibody no. 3).
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Figure 2: Fyn, but not Lck, is required for TCR-stimulated Pyk2 tyrosine phosphorylation. (A) Pyk2 phosphorylation in Jurkat T cells. Left, Jurkat cells were stimulated with anti-TCR (mAb C305) for the indicated period of times. Right, Jurkat cells were stimulated with antiTCR or ionomycin (IONO) for 2 min. Cells were lysed, Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 3), and analyzed by immunoblotting using anti–P-Tyr mAb RC20. The level of Pyk2 in each sample was verified either by immunoblotting an aliquot of anti-Pyk2 immunoprecipitates with antiPyk2 (antibody no. 3) (left) or by stripping and reprobing the same blot with anti-Pyk2 (right). (B) Thymocytes isolated from wildtype (Wt), lck−/−, or fyn−/− mice were either left unstimulated or stimulated with the anti-CD3 mAb for 3 min. Cells were lysed, Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 600), and analyzed by immunoblotting using anti-P-Tyr mAb 4G10. The same blot was stripped and reprobed with antiPyk2 (antibody no. 3).

Mentions: To determine whether Pyk2 is a target for Fyn during TCR signaling, we first tested whether Pyk2 becomes tyrosine phosphorylated upon TCR stimulation. We found that Pyk2 was weakly tyrosine phosphorylated at basal state in T cells. Stimulation of the TCR resulted in a rapid increase in Pyk2 tyrosine phosphorylation in human Jurkat T cells (Fig. 2 A, left) or in normal mouse thymocytes and lymph node T cells (Fig. 2 B). Enhanced tyrosine phosphorylation of Pyk2 is relatively specific to TCR stimulation, as activation via Fas or the costimulator receptor CD28 did not lead to increased Pyk2 tyrosine phosphorylation (data not shown). Furthermore, in contrast with other systems, such as the nicotinic acetylcholine receptor expressed in neuronal cells, which induce Pyk2 tyrosine phosphorylation by stimulation of extracellular calcium influx (17, 20, 24), TCR-induced tyrosine phosphorylation of Pyk2 is Ca2+ independent. Treatment of Jurkat T cells (Fig. 2 A, right) or mouse thymocytes (data not shown) with the calcium ionophore, ionomycin, did not stimulate Pyk2 tyrosine phosphorylation. Moreover, EGTA had no effect on TCRstimulated Pyk2 tyrosine phosphorylation in these cells (data not shown). Thus, Pyk2 tyrosine phosphorylation is likely to be an event proximal to Ca2+ mobilization during TCR signaling. Together, these results demonstrate that Pyk2 is a newly identified physiological target of activated PTK(s) after TCR stimulation.


Tyrosine phosphorylation of Pyk2 is selectively regulated by Fyn during TCR signaling.

Qian D, Lev S, van Oers NS, Dikic I, Schlessinger J, Weiss A - J. Exp. Med. (1997)

Fyn, but not Lck, is  required for TCR-stimulated  Pyk2 tyrosine phosphorylation.  (A) Pyk2 phosphorylation in Jurkat T cells. Left, Jurkat cells were  stimulated with anti-TCR  (mAb C305) for the indicated  period of times. Right, Jurkat  cells were stimulated with antiTCR or ionomycin (IONO) for  2 min. Cells were lysed, Pyk2  was immunoprecipitated with  anti-Pyk2 (antibody no. 3), and  analyzed by immunoblotting using anti–P-Tyr mAb RC20. The  level of Pyk2 in each sample was  verified either by immunoblotting an aliquot of anti-Pyk2 immunoprecipitates with antiPyk2 (antibody no. 3) (left) or by  stripping and reprobing the same  blot with anti-Pyk2 (right). (B)  Thymocytes isolated from wildtype (Wt), lck−/−, or fyn−/− mice  were either left unstimulated or  stimulated with the anti-CD3  mAb for 3 min. Cells were lysed,  Pyk2 was immunoprecipitated  with anti-Pyk2 (antibody no.  600), and analyzed by immunoblotting using anti-P-Tyr mAb  4G10. The same blot was  stripped and reprobed with antiPyk2 (antibody no. 3).
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Figure 2: Fyn, but not Lck, is required for TCR-stimulated Pyk2 tyrosine phosphorylation. (A) Pyk2 phosphorylation in Jurkat T cells. Left, Jurkat cells were stimulated with anti-TCR (mAb C305) for the indicated period of times. Right, Jurkat cells were stimulated with antiTCR or ionomycin (IONO) for 2 min. Cells were lysed, Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 3), and analyzed by immunoblotting using anti–P-Tyr mAb RC20. The level of Pyk2 in each sample was verified either by immunoblotting an aliquot of anti-Pyk2 immunoprecipitates with antiPyk2 (antibody no. 3) (left) or by stripping and reprobing the same blot with anti-Pyk2 (right). (B) Thymocytes isolated from wildtype (Wt), lck−/−, or fyn−/− mice were either left unstimulated or stimulated with the anti-CD3 mAb for 3 min. Cells were lysed, Pyk2 was immunoprecipitated with anti-Pyk2 (antibody no. 600), and analyzed by immunoblotting using anti-P-Tyr mAb 4G10. The same blot was stripped and reprobed with antiPyk2 (antibody no. 3).
Mentions: To determine whether Pyk2 is a target for Fyn during TCR signaling, we first tested whether Pyk2 becomes tyrosine phosphorylated upon TCR stimulation. We found that Pyk2 was weakly tyrosine phosphorylated at basal state in T cells. Stimulation of the TCR resulted in a rapid increase in Pyk2 tyrosine phosphorylation in human Jurkat T cells (Fig. 2 A, left) or in normal mouse thymocytes and lymph node T cells (Fig. 2 B). Enhanced tyrosine phosphorylation of Pyk2 is relatively specific to TCR stimulation, as activation via Fas or the costimulator receptor CD28 did not lead to increased Pyk2 tyrosine phosphorylation (data not shown). Furthermore, in contrast with other systems, such as the nicotinic acetylcholine receptor expressed in neuronal cells, which induce Pyk2 tyrosine phosphorylation by stimulation of extracellular calcium influx (17, 20, 24), TCR-induced tyrosine phosphorylation of Pyk2 is Ca2+ independent. Treatment of Jurkat T cells (Fig. 2 A, right) or mouse thymocytes (data not shown) with the calcium ionophore, ionomycin, did not stimulate Pyk2 tyrosine phosphorylation. Moreover, EGTA had no effect on TCRstimulated Pyk2 tyrosine phosphorylation in these cells (data not shown). Thus, Pyk2 tyrosine phosphorylation is likely to be an event proximal to Ca2+ mobilization during TCR signaling. Together, these results demonstrate that Pyk2 is a newly identified physiological target of activated PTK(s) after TCR stimulation.

Bottom Line: Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation.The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro.Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco 94143, USA.

ABSTRACT
The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-zeta chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.

Show MeSH
Related in: MedlinePlus