![Figure 5. LFA-1– and ADAP-induced actin cloud formation requires SLP-76 and JNK activation. (A) CD8/ADAP induced actin cloud formation in Jurkat mutants lacking ZAP-70 (ZAP-70null) but not SLP-76 (SLP-76null). CD8/ADAP was transfected into Jurkat (E6.1) and Jurkat mutants lacking ZAP-70 (P116) or SLP-76 (J14). The expression levels of CD8/ADAP in these cells were equivalent (not shown). Each CD8+ transfectant was stimulated on coverslips coated with anti-CD8 for 30 minutes. (B) Actin cloud formation requires JNK activation. CD8/ADAP-expressing Jurkat cells were pretreated for 30 minutes with various inhibitors: cytochalasin B for actin rearrangement (Cyt.B), PP2 for src family kinases, wortmannin (Wort) for PI3K, FK506 (FK) for calcineurin, PD98059 (PD) for Mek1, SP600125 (SP) for JNK, and SB203580 (SB) for p38. Pretreated Jurkat cells were stimulated on coverslips coated with anti-CD8 for 30 minutes. The percentage of cells with actin cloud formation among activated (spread) cells is shown. Approximately 80% of the cells were spread. Activated cells (200) were analyzed per inhibitor, and representative results of 3 independent experiments are shown. (C) LFA-1–induced actin cloud formation requires JNK activation. Jurkat cells were pretreated with 3 MAPK inhibitors (SP600125 [JNK], PD98059 [Mek1], and SB203580 [p38]), and stimulated on coverslips coated with anti–LFA-1 for 30 minutes. (D) Quantification of panel C. Data represent the mean ± SD of 400 cells each in 3 experiments. (E) Specific inhibition of actin cloud formation by overexpression of dn-JNK. dn-JNK in a vector containing IRES–rat CD2 or vector alone was transfected into Jurkat cells and stimulated on coverslips coated with anti–LFA-1. Rat CD2 was stained green and actin was stained red (right panel). High and low expressions of dn-JNK was determined from the relative brightness of rat CD2 (left panel). Cells (200) were analyzed for panels B, D, and E. (F) Activation of JNK upon LFA-1 stimulation. Jurkat cells were stimulated in plates coated with 10 mg/mL anti–LFA-1 or anti-CD3 for 30 minutes. Total cell lysates were blotted with anti–phospho–c-Jun or anti-JNK Ab. White arrows indicate representative actin clouds.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/1/10.1182_blood-2005-12-020164/4/zh80010705680005.jpeg?Expires=1735537696&Signature=UdQFl7Cv7Jxo0q6FdNd8eXEBlPw1rYWRorZzMmXstAkmgF9hu98FSmwEdho4QzBDdOqrfNcXpcgNId8IuOGbjCw~OkNtg9GWuGfvV4MBuTO5bVdNdjEZaY7Rg23lMgFfbunVJa4l9A8u6H7AODEAIAOaIuEdYMj~OrwhD9t7ZBhMBVPzAqrfxK9gIieAxQEIVvSKePwBXintDRt~icml1HldKUKhCt8oRenL1UQq3n-O2XxeLHQCffE-rmgRzetAWAy4emo7NNYqKP1Wzlfnhe4M4c0XO3xep09ZdSHMLCIG0iwReMSNd41ZzxUBJui8a5RqZ9-Qpc5-xHZ83z3b0A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
LFA-1– and ADAP-induced actin cloud formation requires SLP-76 and JNK activation. (A) CD8/ADAP induced actin cloud formation in Jurkat mutants lacking ZAP-70 (ZAP-70null) but not SLP-76 (SLP-76null). CD8/ADAP was transfected into Jurkat (E6.1) and Jurkat mutants lacking ZAP-70 (P116) or SLP-76 (J14). The expression levels of CD8/ADAP in these cells were equivalent (not shown). Each CD8+ transfectant was stimulated on coverslips coated with anti-CD8 for 30 minutes. (B) Actin cloud formation requires JNK activation. CD8/ADAP-expressing Jurkat cells were pretreated for 30 minutes with various inhibitors: cytochalasin B for actin rearrangement (Cyt.B), PP2 for src family kinases, wortmannin (Wort) for PI3K, FK506 (FK) for calcineurin, PD98059 (PD) for Mek1, SP600125 (SP) for JNK, and SB203580 (SB) for p38. Pretreated Jurkat cells were stimulated on coverslips coated with anti-CD8 for 30 minutes. The percentage of cells with actin cloud formation among activated (spread) cells is shown. Approximately 80% of the cells were spread. Activated cells (200) were analyzed per inhibitor, and representative results of 3 independent experiments are shown. (C) LFA-1–induced actin cloud formation requires JNK activation. Jurkat cells were pretreated with 3 MAPK inhibitors (SP600125 [JNK], PD98059 [Mek1], and SB203580 [p38]), and stimulated on coverslips coated with anti–LFA-1 for 30 minutes. (D) Quantification of panel C. Data represent the mean ± SD of 400 cells each in 3 experiments. (E) Specific inhibition of actin cloud formation by overexpression of dn-JNK. dn-JNK in a vector containing IRES–rat CD2 or vector alone was transfected into Jurkat cells and stimulated on coverslips coated with anti–LFA-1. Rat CD2 was stained green and actin was stained red (right panel). High and low expressions of dn-JNK was determined from the relative brightness of rat CD2 (left panel). Cells (200) were analyzed for panels B, D, and E. (F) Activation of JNK upon LFA-1 stimulation. Jurkat cells were stimulated in plates coated with 10 mg/mL anti–LFA-1 or anti-CD3 for 30 minutes. Total cell lysates were blotted with anti–phospho–c-Jun or anti-JNK Ab. White arrows indicate representative actin clouds.
