TY - JOUR
T1 - Phosphorylation at serine 318 is not required for inhibition of T cell activation by ALX
AU - Shapiro, Michael J.
AU - Spruce, Lynn
AU - Sundsbak, Rhianna
AU - Thapa, Puspa
AU - Shapiro, Virginia Smith
N1 - Funding Information:
We would like to thank Steve Seeholzer and Mike Rosenblatt in the Children’s Hospital of Philadelphia Protein and Proteomics core, and Art Weiss for the C305 antibody. The authors declare that they have no competing/financial conflicts of interest. This work was supported by NIH R01 AI054974 to V.S.S.
PY - 2010/6/11
Y1 - 2010/6/11
N2 - The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation.
AB - The activation of T cells and the initiation of an immune response is tightly controlled by both positive and negative regulators. Two adaptors which function as negative regulators of T cell activation are ALX and LAX. ALX constitutively associates with LAX in T cells, and T cells from mice deficient in ALX and LAX display similar hyper-responsiveness upon T cell receptor (TCR)/CD28 stimulation, including increased production of interleukin-2. During T cell activation, ALX is inducibly phosphorylated, however the site of ALX phosphorylation had not been previously identified and the role of phosphorylation in the inhibitory function of ALX was not known. Here, using mass spectrometry, we demonstrate that ALX is phosphorylated on a serine at position 318. Substitution of alanine for serine at this position (ALX S318A) leads to an abrogation of the mobility shift in ALX induced upon TCR/CD28 stimulation. However, ALX S318A retained the ability to bind to and stimulate tyrosine phosphorylation of LAX. In addition, overexpression of ALX S318A inhibited RE/AP activation upon TCR/CD28 stimulation to a similar extent as wild-type ALX. Therefore, although ALX is inducibly phosphorylated upon TCR/CD28 stimulation, this phosphorylation is not required for ALX to inhibit T cell activation.
KW - ALX
KW - Adaptor
KW - Phosphorylation
KW - T cell
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U2 - 10.1016/j.bbrc.2010.05.043
DO - 10.1016/j.bbrc.2010.05.043
M3 - Article
C2 - 20471366
AN - SCOPUS:77953288350
SN - 0006-291X
VL - 396
SP - 994
EP - 998
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -