Abstract
T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. However, when unregulated these same pathways promote pathologies ranging from autoimmune disorders to cancer. While the functional role of Interleukin-2 and downstream effector molecules is relatively clear, the repertoire of phosphoregulatory proteins downstream of this pathway is incomplete. To identify phosphoproteins downstream of common gamma chain receptor, YT cells were radiolabeled with [32P]-orthophosphate and stimulated with Interleukin-2. Subsequently, tyrosine phosphorylated proteins were immunopurified and subjected to tandem mass spectrometry—leading to the identification of CrkL. Phosphoamino acid analysis revealed concurrent serine phosphorylation of CrkL and was later identified as S114 by mass spectrometry analysis. S114 was inducible through stimulation with Interleukin-2 or T-cell receptor stimulation. Polyclonal antibodies were generated against CrkL phospho-S114, and used to show its inducibility by multiple stimuli. These findings confirm CrkL as an Interleukin-2 responsive protein that becomes phosphorylated at S114 by a kinase/s downstream of PI3K and MEK/ERK signaling.
Highlights
T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity
The resulting IL-2 induced tyrosine phosphorylated proteins were assessed by mass spectrometry and phosphoamino acid analysis resulted in the detection of a 37 kDa protein identified as -Crk-like protein (CrkL)
Phosphorylation of CrkL S114 was dependent on serine/threonine kinases downstream of Phosphoinositide 3-Kinase (PI3K) and MAP Kinase (MAPK)/ Extracellular Signal-Regulated Kinase (ERK) pathways that are induced by IL-2
Summary
T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. Dysfunction in cell signaling can lead to the manifestation of immunological disorders such as immunodeficiency, autoimmunity and hematopoietic m alignancies[1,2,3] Many of these conditions are the result of abnormal regulation of the common gamma chain (γc) family of cytokines[2,4,5,6] involved in T-cell and natural killer (NK) cell function. While the functional role of IL-2 and downstream effector molecules is relatively clear, promoting differentiation, proliferation, and survival of T cells, the various co-regulatory pathways involved in this cascade are much less understood To better understand this signaling network, we sought to identify novel IL-2 phospho-Tyrosine (pY) inducible proteins. Phosphorylation of CrkL S114 was dependent on serine/threonine kinases downstream of PI3K and MAPK/ ERK pathways that are induced by IL-2
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