ZAP-70 Enhances IgM Signaling in CLL, Apart from Kinase Activity

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[585] ZAP-70 Enhances IgM Signaling in Chronic Lymphocytic Leukemia

Cells Independent of Its Tyrosine Kinase Activity. Session Type: Oral

Session

Liguang Chen, Lang Huynh, Arthur Weiss, J. Kipps s UCSD

Cancer Center, University of California, San Diego, La Jolla, CA,

USA; Department of Medicine, Medical Institute, San

Francisco, University of California, San Francisco, San Francisco,

CA, USA

We found that expression of the zeta-associated protein of 70 kD (ZAP-

70) by chronic lymphocytic leukemia (CLL) B cells enhanced IgM-

receptor signaling, even though such CLL cells also expressed a

highly-related and more active tyrosine kinase, p72Syk.

To investigate whether the kinase activity of ZAP-70 is necessary for

this effect, we transfected ZAP-70-negative primary leukemia cells

with expression vectors encoding either wild-type ZAP-70 or ZAP-70-

KI, a mutant ZAP-70 that has an inactivating point mutation in the

ATP-binding site (Lys369-Ala) and that lacks tyrosine kinase

activity. We achieved high-level expression of ZAP-70 or ZAP-70-KI in

transfected CLL cell samples and compared these cells with each other

and with the same CLL cell samples that had been mock transfected

with a control vector and that remained negative for expression of

ZAP-70 (n = 7).

To examine the B-cell-receptor (BCR) signaling potential of these

cells we assessed the extent of tyrosine phosphorylation of p72Syk, B-

cell linker protein (BLNK), and phospholipase C gamma (PLC-), and

measured intracellular calcium flux ([Ca2+]I) before and 5-10 minutes

after surface IgM ligation with F(ab)2 anti- antibody. Treatment of

control mock-transfected ZAP-70-negative CLL cells with anti-

resulted in negligible-to-minimal increases in phosphorylation of

these cytosolic proteins, with mean increases in phosphorylation of

p72Syk, BLNK, and PLC- of only 46% 47% (S.D), 173% 112%, and 73%

76%, respectively. CLL cells engineered to express non-mutated ZAP-70

experienced significantly higher levels of protein tyrosine

phosphorylation following treatment with anti-, with mean increases

in phosphorylation of p72Syk, BLNK, and PLC- of 186% 102%, 490%

323%, and 722% 836%, respectively, as had been noted in earlier

studies. Surprisingly, CLL cells made to express the tyrosine-kinase-

defective mutant ZAP-70KI also experienced significantly higher

levels of protein tyrosine phosphorylation following treatment with

anti- than did the mock-transfected CLL cells, with mean increases in

phosphorylation of p72Syk, BLNK, and PLC- of 172% 94%, 431% 261%,

and 759% 637%, respectively. These values were similar to those

noted for anti- treated CLL cells that expressed the wild-type ZAP-70

protein. These results were reflected also in the ([Ca2+]I) induced

by anti- in each of the three groups of CLL cells. Whereas anti-

treatment of mock-transfected CLL cells resulted in negligible-to-

minimal increases in ([Ca2+]I) of 0.25 units 0.19, anti- treatment

of ZAP-70-transfected or ZAP-70KI-transfected CLL cells resulted in

increases in ([Ca2+]I) of 1.05 units 0.53 and 0.95 units 0.46,

respectively.

These values each were significantly higher than that noted for mock-

transfected CLL cells. We conclude that the tyrosine-kinase activity

of ZAP-70 is not required for ZAP-70 to enhance BCR signaling in CLL

cells.