ZAP-70 Does Not Undergo Full Activation in CLL Cells

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[178] ZAP-70 Is Not Phosphorylated on the Activating Tyrosine

Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma

Cells. Session Type: Oral Session

Stefania Gobessi, Aleksandar Petlickovski, Luca ti, Dimitar G.

Efremov ICGEB Hematology Group - Monterotondo Outstation, CNR

Campus " A. Buzzati Traverso " , Rome, Italy; Hematology Institute,

Catholic University Hospital " A. Gemelli " , Rome, Italy

The protein tyrosine kinase ZAP-70 is expressed at high levels in

leukemic B-cells from chronic lymphocytic leukemia (CLL) patients

with progressive disease and short survival. ZAP-70 is a key

component of the proximal T-cell receptor signaling pathway and is

highly homologous to Syk, an important B-cell receptor signaling

(BCR) molecule.

Recent studies indicate that ZAP-70 may participate in BCR signaling

as well, but the mechanism of action is still not well understood.

In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on

Tyr319 by the Src-like kinase Lck, which results in the release of

the ZAP-70 kinase domain from an autoinhibited state to a fully

active conformation. The Tyr319 site in ZAP-70 corresponds to the

Tyr352 site in Syk, which is phosphorylated in B-cells following BCR

stimulation.

We therefore investigated the activation status of ZAP-70 and Syk in

BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and

Tyr352 as markers of their activation. Analysis of 10 ZAP-70-

positive CLL samples by immunoblotting with the phospho-

ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not

phosphorylated at this site either before or after BCR stimulation,

although in control experiments with Jurkat T-cells ZAP-70 became

phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352

site in Syk was phosphorylated following BCR stimulation in 6 of the

10 CLL B-cell samples.

To further investigate the reasons for the unexpected lack of ZAP-70

activation in CLL B-cells, we produced stable transfectants of the

BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to

those found in CLL cases with progressive disease. In agreement with

the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not

phosphorylated either before or after BCR stimulation.

Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and

this kinase is expressed also in CLL B-cells, we ectopically

expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319

site was not phosphorylated, indicating that ZAP-70 does not undergo

activation of the kinase domain also in this cellular system.

In contrast, BCR crosslinking in BJAB cells induced significant

phosphorylation of Tyr352 in Syk, which was further enhanced in the

clones that coexpressed ZAP-70. Furthermore, analysis of downstream

signaling pathways following BCR stimulation showed stronger and

prolonged activation of ERK and to a lesser extent Akt in the ZAP-70

positive clones, whereas no difference was observed in terms of

activation of PLC-ã2, JNK and degradation of the NF-kB inhibitor

IkB.

These data indicate that ZAP-70 does not undergo full activation in

B-cells, but can still enhance activation of certain downstream BCR

signaling pathways, possibly by affecting the activity of the

related PTK Syk.