Abstract
While the immune cell adaptor protein SKAP1 mediates LFA-1 activation induced by antigen-receptor (TCR/CD3) ligation on T-cells, it is unclear whether the adaptor interacts with other mediators of T-cell function. In this context, the serine/threonine kinase, polo-like kinase (PLK1) regulates multiple steps in the mitotic and cell cycle progression of mammalian cells. Here, we show that SKAP1 is phosphorylated by and binds to PLK1 for the optimal cycling of T-cells. PLK1 binds to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity. Further, siRNA knock-down of SKAP1 reduced the rate of T-cell division concurrent with a delay in the expression of PLK1, Cyclin A and pH3. Reconstitution of these KD cells with WT SKAP1, but not the SKAP1 S31 mutant, restored normal cell division. SKAP1-PLK1 binding is dynamically regulated during the cell cycle of T-cells. Our findings identify a novel role for SKAP1 in the regulation of PLK1 and optimal cell cycling needed for T-cell clonal expansion in response to antigenic activation.
Highlights
Polo-like kinase 1 (PLK1) is a serine/threonine kinase that regulates the mitosis of mammalian cells
Purified GSTSKAP1 was added to precipitates of individual kinases that included CDK1, CDK2, MAPK, Aurora B, CAMK, PLK3, PLK1, MST1 and ZAP-70 in the presence of radio-active phosphate followed by an assessment of phosphorylation on SDS-PAGE
We define a second function for SKAP1 involving the regulation of PLK1 activity and T-cell cycling
Summary
Polo-like kinase 1 (PLK1) is a serine/threonine kinase that regulates the mitosis of mammalian cells. There are five different PLK family members expressed in mammalian cells where they regulate the cell cycle[2]. SLP-76 (SH2 domain containing leukocyte protein of 76 kDa) is one such adaptor that is needed for phospholipase Cγ1 (PLCγ1) activation, calcium mobilization and thymic differentiation[25,26] It has an N-terminal sterile-α motif (SAM) and a carboxy-terminal SH2 domain that binds to adhesion and degranulation-promoting adapter protein (ADAP)[27,28,29,30,31] and the hematopoietic progenitor kinase-1 (HPK-1)[31]. We show that PLK1 phosphorylates and binds to SKAP1, an interaction that is cell cycle dependent during mitosis. Our findings identify a novel role for SKAP1 as a scaffold for PLK1 in the regulation for the cell cycle of T-cells
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