The effect of serine 59 phosphorylation on the conformation of the protein binding domains of the protein tyrosine kinase Lck

Meredith B Baker, Purdue University

Abstract

The protein tyrosine kinase Lck is a vital member of signaling cascades initiated by antigen stimulation of the TCR. Much of the knowledge about the regulation of Lck is based on several structural studies of the individual domains of Lck and computational models of other family members. However, there are no structures of the complete unique domain of Lck, a 60 residue region N-terminal to the SH3 domain that is sequentially unlike other family members. The unique region of Lck contains two major phosphorylation sites, serine 42 and serine 59, phosphorylated by PKA and Erk MAPK, respectively. Previous studies have shown that the phosphorylation of Lck serine 59 caused a reduction in mobility of Lck in SDS-PAGE gels and prevented the binding of the tyrosine phosphatase SHP-1 to Lck. We postulated that the unique region of Lck interacted with the SH3 and SH2 domains of Lck and the phosphorylation of serine 59 altered the conformation of these protein binding domains of Lck. We designed and expressed wild-type and S59D protein constructs of the N-terminal half of Lck for structural studies. We analyzed these constructs using analytical ultracentrifugation and nuclear magnetic resonance. Our data show that the overall shape of the Lck protein was not altered in the presence of the serine phosphorylation mimic. Although the overall conformation of the Lck protein was not dramatically changed, we identified several residues surrounding the mutation site, in the connector region, and in the SH3 and SH2 domains that had an altered orientation in the presence of the phosphorylation mimic. The altered orientation of these residues may change the specificity of the SH3 or SH2 domain of Lck. We identified two proteins SHP-1 and SKAP55 that bound to Lck only in the absence of serine 59 phosphorylation. These data suggest that the phosphorylation of serine 59 may create a new binding motif for Lck binding partners, affect the signaling pathways in which Lck participates by altering the binding capabilities of potential binding partners, or provide a control mechanism for Lck activity.

Degree

Ph.D.

Advisors

Harrison, Purdue University.

Subject Area

Molecular biology|Cellular biology

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