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Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

Vang T, Torgersen KM, Sundvold V, Saxena M, Levy FO, Skålhegg BS, Hansson V, Mustelin T, Taskén K - J. Exp. Med. (2001)

Bottom Line: Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation.Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin.We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway.

ABSTRACT
In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

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PKA phosphorylation increases the tyrosine kinase activity of Csk towards an endogenous substrate. Tyrosine phosphorylation of heat-inactivated (65°C for 10 min) purified Lck enzyme (30 ng/μl, cat. no. 14-106; Upstate Biotechnology) by Csk (0.3 ng/μl) was assessed either in the presence or absence of PKA catalytic subunit Cα (10 ng/μl) in a buffer containing 5 mM Mg2+ and 200 μM ATP at 30°C for 10 min. Reactions were stopped by the addition of SDS sample buffer, subjected to SDS-PAGE, and phosphotyrosine content of Lck was assessed by antiphosphotyrosine immunoblotting (4G10). Densitometric scanning was performed to evaluate the level of Csk-mediated tyrosine phosphorylation of Lck in the absence and presence of PKA. n.d., not done.
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Figure 4: PKA phosphorylation increases the tyrosine kinase activity of Csk towards an endogenous substrate. Tyrosine phosphorylation of heat-inactivated (65°C for 10 min) purified Lck enzyme (30 ng/μl, cat. no. 14-106; Upstate Biotechnology) by Csk (0.3 ng/μl) was assessed either in the presence or absence of PKA catalytic subunit Cα (10 ng/μl) in a buffer containing 5 mM Mg2+ and 200 μM ATP at 30°C for 10 min. Reactions were stopped by the addition of SDS sample buffer, subjected to SDS-PAGE, and phosphotyrosine content of Lck was assessed by antiphosphotyrosine immunoblotting (4G10). Densitometric scanning was performed to evaluate the level of Csk-mediated tyrosine phosphorylation of Lck in the absence and presence of PKA. n.d., not done.

Mentions: To look at a normal substrate for Csk, heat-inactivated Lck was used as substrate and the activity of Csk in the presence and absence of PKA was examined. When Csk was limiting in the reaction, Csk-mediated tyrosine phosphorylation of Lck was 4.8-fold stronger in the presence than in the absence of PKA (Fig. 4).


Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor.

Vang T, Torgersen KM, Sundvold V, Saxena M, Levy FO, Skålhegg BS, Hansson V, Mustelin T, Taskén K - J. Exp. Med. (2001)

PKA phosphorylation increases the tyrosine kinase activity of Csk towards an endogenous substrate. Tyrosine phosphorylation of heat-inactivated (65°C for 10 min) purified Lck enzyme (30 ng/μl, cat. no. 14-106; Upstate Biotechnology) by Csk (0.3 ng/μl) was assessed either in the presence or absence of PKA catalytic subunit Cα (10 ng/μl) in a buffer containing 5 mM Mg2+ and 200 μM ATP at 30°C for 10 min. Reactions were stopped by the addition of SDS sample buffer, subjected to SDS-PAGE, and phosphotyrosine content of Lck was assessed by antiphosphotyrosine immunoblotting (4G10). Densitometric scanning was performed to evaluate the level of Csk-mediated tyrosine phosphorylation of Lck in the absence and presence of PKA. n.d., not done.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195911&req=5

Figure 4: PKA phosphorylation increases the tyrosine kinase activity of Csk towards an endogenous substrate. Tyrosine phosphorylation of heat-inactivated (65°C for 10 min) purified Lck enzyme (30 ng/μl, cat. no. 14-106; Upstate Biotechnology) by Csk (0.3 ng/μl) was assessed either in the presence or absence of PKA catalytic subunit Cα (10 ng/μl) in a buffer containing 5 mM Mg2+ and 200 μM ATP at 30°C for 10 min. Reactions were stopped by the addition of SDS sample buffer, subjected to SDS-PAGE, and phosphotyrosine content of Lck was assessed by antiphosphotyrosine immunoblotting (4G10). Densitometric scanning was performed to evaluate the level of Csk-mediated tyrosine phosphorylation of Lck in the absence and presence of PKA. n.d., not done.
Mentions: To look at a normal substrate for Csk, heat-inactivated Lck was used as substrate and the activity of Csk in the presence and absence of PKA was examined. When Csk was limiting in the reaction, Csk-mediated tyrosine phosphorylation of Lck was 4.8-fold stronger in the presence than in the absence of PKA (Fig. 4).

Bottom Line: Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation.Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin.We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway.

ABSTRACT
In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.

Show MeSH
Related in: MedlinePlus