Comment on "PDK1 Nucleates T Cell Receptor-Induced Signaling Complex for NF-
B Activation"
Thomas Gruber1,
Michael Freeley2*,
Nikolaus Thuille1,
Isabelle Heit3,
Stephen Shaw4,
Aideen Long2* and
Gottfried Baier1
1 Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Austria.
2 Department of Biochemistry, Royal College of Surgeons, Dublin, Ireland.
3 AltanaPharma, Konstanz, Germany.
4 Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
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Fig. 1. (A) Constitutive activation-loop phosphorylation of PKC in CD3+ T cells. Murine CD3+ T cells were negatively selected and purified from lymph nodes and spleen of wild-type and PKC–/– mice. PKC was immunoprecipitated (IP) from resting (–) or 1-hour stimulated (+; by solid-phase antibodies to CD3 and CD28) mouse CD3+ cells, employing the PKC-specific pAb from Santa Cruz. The Thr-538 phosphostatus was determined in an immunoblot, employing the PKC (p)Thr-538 specific pAb from CST. (B) Phosphopeptide antibody to Thr-538 reacts in an epitope-specific manner. Wild-type and T538A mutant PKC proteins were expressed in baculovirus and purified to homogeneity and tested in vitro with the (p)Thr-538 PKC antiserum from CST. Immunoreactivity was phosphosite-specific for Thr-538 of PKC as no immunoreactivity was observed with the PKC T538A mutant protein.
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Fig. 2. Constitutive activation-loop phosphorylation of PKC in Jurkat T cells. Jurkat cells were maintained in 10% FCS (A) or 0.5% FCS for 48 hours (B) and then transiently transfected with wild-type PKC and T538A mutant. Serum-starved cells were also transfected with a GFP inert protein control and maintained in 0.5% FCS at all times. After 24 hours, cells were activated with solid-phase antibodies to CD3 and CD28 for various times, as indicated. The Thr-538 phospho-status was determined by immunoblotting of whole cell extracts (WCE). As a loading control, the membranes were stripped and reprobed with antibodies to total PKC. A nonspecific, albeit inducible 72-kD protein band recognized by the (p)Thr-538 antiserum from CST is marked with an asterisk. As an internal activation marker, the lysates were immunostained in parallel with an antibody to phospho-MAPK that detects dually phosphorylated (active) extracellular signal–regulated kinase 1 (ERK1) and ERK2.
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Fig. 3. Constitutive activation-loop phosphorylation of endogenous PKC in Jurkat T cells. Cells were maintained in 10% FCS at all times and stimulated with solid-phase antibodies to CD3 and CD28 for various times, as indicated. Thr-538 phospho-status determination, loading control, and internal activation marker were the same as described in Fig. 2.
[View Larger Version of this Image (25K GIF file)]
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