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. 2007 Mar 19;204(3):681–691. doi: 10.1084/jem.20062066

Figure 6.

Figure 6.

HPK-1 phosphorylates SLP-76 at serine 376. (A) Knockdown of HPK-1 expression in Jurkat T cells was performed by transient transfection of synthetic siRNAs. Cells were electroporated in the presence of medium alone (mock), control siRNAs (ctrl), or HPK-1–specific siRNAs (HPK1). After 72 h, cells were left unstimulated or activated by anti-CD3 mAb, and lysates were fractionated on Nu-PAGE gels. HPK-1 expression was assessed by anti–HPK-1 immunoblotting (top row). Phosphorylation of SLP-76 and PLC-γ1 was then assessed by immunoblotting with anti-pS376 (second row from top), anti-pY128 (third row from top), and anti–PLC-γ1 pY783 (fourth row from top). Equal protein loading in each lane was demonstrated by anti–SLP-76 and anti–14-3-3ζ immunoblotting (two bottom rows). (B) J14-WT cells were transiently transfected with either control siRNAs (ctrl) or HPK-1–specific siRNAs (HPK1) as in A. After 72 h, cells were left unstimulated or activated by anti-CD3 mAb for 10 min, and lysates were immunoprecipitated by anti-FLAG antibodies. Aliquots of the lysate were analyzed by immunoblotting with anti–HPK-1 or anti–SLP-76 antibodies (first and second row from top, respectively). Immunoprecipitates were immunoblotted with anti–SLP-76 (third row), anti-pS376 (fourth row), and anti–14-3-3ζ (bottom row). Quantification of band intensities and normalization by the amount of SLP-76 in each lane indicated that HPK-1 expression, pS376 phosphorylation, and 14-3-3ζ binding to SLP-76 were reduced in this experiment by 68, 58, and 50%, respectively. (C) COS-7 cells were transfected with FLAG–SLP-76 or HPK-1–HA constructs, WT, or a kinase-defective mutant (KD). After 36 h, cells were harvested, lysed, and immunoprecipitated with anti-FLAG (FLAG–SLP-76 transfection) or anti-HA antibodies (HPK-1 WT– or KD-transfected cells). FLAG–SLP-76 was eluted and mixed with bead-bound HPK-1–WT or –KD. Samples were then incubated for 30 min at 37°C in the presence or absence of ATP. Protein mixtures were analyzed by immunoblotting with anti-FLAG (top row), anti-HA (middle row), and anti-pS376 antibodies (bottom row).