Wiskott-Aldrich Syndrome causing mutation, Pro373Ser restricts conformational changes essential for WASP activity in T-cells.
Wiskott-Aldrich Syndrome (WAS) is caused by mutations in Wiskott-Aldrich Syndrome Protein (WASP) and majority of the mutations are found in the WASP Homology 1 (WH1) domain which mediates interaction with WIP (WASP Interacting Protein), a WASP chaperone. Two point mutations together in the proline rich region (PRR) domain of WASP (S339Y/P373S) have been reported to cause WAS however the molecular defect has not been characterized. Expression of these mutants separately (WASPR(S339Y), WASPR(P373S)) or together (WASPR(SP/YS)) did not rescue the chemotaxis defect or membrane projection defect of Jurkat(WKD) T-cells (WASP knockdown). This is not due to the inability of WASP-PRR mutants to form functional WASP-WIP complex in growth rescue experiments in las17Δ yeast strain. Expression of WASPR(S339Y) but not WASPR(P373S) or WASPR(SP/YS) rescued the IL-2 expression defect of Jurkat(WKD) T-cells, suggesting that Pro373Ser mutation alone is sufficient to inhibit WASP functions in T-cell activation. The diffused localization of WASP-PRR mutants in activated Jurkat T-cells suggests that Ser339 and Pro373 are critical for WASP localization. WASP-PRR mutations either together or individually did not abolish interaction of WASP with sixteen WASP binding proteins including Hck, however they caused reduction in Hck mediated tyrosine phosphorylation of WASP which is critical for WASP activity. The auto-inhibitory conformation of WASP(P373S) mutant was not relieved by the binding of Toca-1 or Nck1. Thus, our results suggest that Pro373Ser mutation reduces Tyr291 phosphorylation and prevents conformational changes required for WASP activity in chemotaxis and T-cell activation. Thus Pro3373Ser is probably responsible for all the defects associated with WAS in the patients.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
Neeraj
N
Jain
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
Bhawana
B
George
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore. Electronic address: thirumaran@ntu.edu.sg.
Thirumaran
T
Thanabalu
D002352:Carrier Proteins / Q000502:physiology; D002634:Chemotaxis, Leukocyte; D006801:Humans; D054992:Immunological Synapses; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D013601:T-Lymphocytes / Q000276:immunology*; D014923:Wiskott-Aldrich Syndrome / Q000235:genetics*; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry / Q000235:genetics*
D002352:Carrier Proteins; C482527:FNBP1L protein, human; D007376:Interleukin-2; D051316:Wiskott-Aldrich Syndrome Protein
Actin cytoskeleton; Cell migration; IL-2 expression; Immunodeficiency; T-cell activation