Characterization of the in Vivo Sites of Serine Phosphorylation on Lck Identifying Serine 59 as a Site of Mitotic Phosphorylation

Kamala P Kesavan,Christina C Isaacson,Curtis L Ashendel,Robert L Geahlen,Marietta L Harrison

doi:10.1074/jbc.m111911200

Kamala P Kesavan, Christina C Isaacson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111911200

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Abstract

The lymphocyte-specific protein-tyrosine kinase Lck plays a critical role in T cell activation. In response to T cell antigen receptor binding Lck undergoes phosphorylation on serine residues that include serines 59 and 194. Serine 59 is phosphorylated by ERK mitogen-activated protein kinase. Recently, we showed that in mitotic T cells Lck becomes hyper-phosphorylated on serine residues. In this report, using one-dimensional phosphopeptide mapping analysis, we identify serine 59 as a site of in vivo mitotic phosphorylation in Lck. The mitotic phosphorylation of serine 59 did not require either the catalytic activity or functional SH2 or SH3 domains of Lck. In addition, the presence of ZAP-70 also was dispensable for the phosphorylation of serine 59. Although previous studies demonstrated that serine 59 is a substrate for the ERK MAPK pathway, inhibitors of this pathway did not block the mitotic phosphorylation of serine 59. These results identify serine 59 as a site of mitotic phosphorylation in Lck and suggest that a pathway distinct from that induced by antigen receptor signaling is responsible for its phosphorylation. Thus, the phosphorylation of serine 59 is the result of two distinct signaling pathways, differentially activated in response to the physiological state of the T cell.

Highlights

Lck is a member of the Src family of non-receptor protein kinases
It was reported that in mature T lymphocytes, Lck was required for progression through the G2-M transition [32] and that cells treated with a Src family selective tyrosine kinase inhibitor were blocked in mitosis [35]
As we reported previously [28], Jurkat cells arrested at mitosis by treatment with the microtubule inhibitor nocodazole contained an additional Lck band which migrated with slower electrophoretic mobility on SDS-polyacrylamide gels (Fig. 1A, lane 2)

Summary

Introduction

Lck is a member of the Src family of non-receptor protein kinases. It primarily is expressed in T lymphocytes and natural killer cells and plays a critical role in T cell development and activation [1,2,3,4,5,6]. In response to T cell stimulation by the ligation of either the T cell antigen receptor (TCR) [13,14,15,16,17,18], the interleukin-2 receptor [19], or treatment with phorbol esters [17, 20, 21], Lck undergoes phosphorylation on amino-terminal serine residues. It was reported that in mature T lymphocytes, Lck was required for progression through the G2-M transition [32] and that cells treated with a Src family selective tyrosine kinase inhibitor were blocked in mitosis [35]. The presence of ZAP-70 was dispensable for the

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