The Mitogen-Activated Protein Kinase Scaffold KSR1 Is Required for Recruitment of Extracellular Signal-Regulated Kinase to the Immunological Synapse (cid:1)

KSR1 is a mitogen-activated protein (MAP) kinase scaffold that enhances the activation of the MAP kinase extracellular signal-regulated kinase (ERK). The function of KSR1 in NK cell function is not known. Here we show that KSR1 is required for efficient NK-mediated cytolysis and polarization of cytolytic granules. Single- cell analysis showed that ERK is activated in an all-or-none fashion in both wild-type and KSR1-deficient cells. In the absence of KSR1, however, the efficiency of ERK activation is attenuated. Imaging studies showed that KSR1 is recruited to the immunological synapse during T-cell activation and that membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse. Kinase suppressor of Ras was originally identified in Drosophila melanogaster (53) and Caenorhabditis elegans (19, 32, 52) as a positive regulator of the extracellular signal-regulated and grown in hIL-2-containing medium (5). Polyclonal rabbit anti-Grb2, rabbit anti-ERK2, rabbit anti-KSR1, and mouse anti-Lck were obtained from Santa Cruz Biotechnology. Polyclonal rabbit anti-phospho-ERK [pERK1/2 (Thr202/Tyr204)] and rabbit anti-phospho-* YFP, respectively. The integrity of all constructs was verified by automated sequencing. For lentivirus production, subconfluent cultures of 293T cells were transfected with packaging plasmid (pHR (cid:4) 8.2 (cid:7) R/pCMV-VSV-G at a ratio of 8:1) and pFLRu-derived plasmid (Y. Feng, H. Zhao, B. Wang, and G. D. Longmore, submitted for publication) using Lipofectamine 2000. The lentivirus infection of Jurkat cells was performed using the protocol described above, and cells expressing the same level of YFP were sorted 3 to 5 days later. immunofluorescence To study of phosphorylated immunological synapse, wild-type (cid:2) carboxyfluorescein succinimidyl centrifugation, resuspended poly- -lysine- glass aspiration medium,

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