Abstract
Wiskott-Aldrich Syndrome protein (WASP) integrates cell signaling pathways to the actin cytoskeleton, which play a critical role in T-cell activation and migration. Hematopoietic cells express both WASP and neural-WASP (N-WASP) which share similar domain structure, yet WASP deficiency causes Wiskott-Aldrich syndrome, suggesting that N-WASP present in the cells is not able to carry out all the functions of WASP. We have identified a unique internal thirty amino acid region (I30) in WASP, which regulates its function in chemotaxis of Jurkat T-cells. Deletion of the I30 region altered the WASP’s closed conformation and impaired its ability to rescue the chemotactic defect of WASP-deficient (JurkatWKD) T-cells. Expression of N-WASP in JurkatWKD T-cells using WASP promoter restored the migration velocity without correcting the chemotactic defect. However, insertion of I30 region in N-WASP (N-WASP-I30) enabled N-WASP to rescue the chemotactic defect of JurkatWKD T-cells. N-WASP-I30-EGFP displayed a punctate localization in contrast to the predominant nuclear localization of N-WASP-EGFP. Thus, our study has demonstrated that the I30 region of WASP is critical for localization and chemotaxis. This suggests that N-WASP’s failure to compensate for WASP in rescuing chemotaxis could be due to the absence of this I30 region.
Highlights
Wiskott Aldrich Syndrome protein (WASP), Neural-Wiskott-Aldrich Syndrome protein (WASP) (N-WASP) and WASP family Verprolinehomologue protein (WAVE) 1, 2, 3 are scaffold proteins that link cell surface signals to actin cytoskeleton[1,2]
The mRNA level of WASP in these hematopoietic cell lines was ~50% higher than that of N-WASP mRNA and negligible WASP mRNA was observed in control cell line HEK293T cells (Fig. 1A)
We found that 40.7% of JurkatWKD T-cells(N-WASP-RFP) migrated to the lower well compared to 57.10% of JurkatWKD T-cells(WASPR-RFP), suggesting that N-WASP is unable to rescue the chemotaxis defect of JurkatWKD T-cells towards SDF-1α (Fig. 2D)
Summary
Wiskott Aldrich Syndrome protein (WASP), Neural-WASP (N-WASP) and WASP family Verprolinehomologue protein (WAVE) 1, 2, 3 are scaffold proteins that link cell surface signals to actin cytoskeleton[1,2]. WASP and N-WASP share similar protein domain organization, which comprises the WASP homology domain (WH1) at their N-terminus followed by a basic region (BR), GTPase binding domain (GBD), a proline rich region (PRR), and a verproline-cofilin-acidic region (VCA) at C-terminus[1]. The VCA region of WASP family proteins interacts with G-actin and promotes actin polymerization by activating the Arp2/3 complex[8,9]. WASP and N-WASP share more than 50% sequence homology, having similar binding partners and comparable basic functions. In resting cells, both WASP and N-WASP exist in an auto-inhibitory closed conformation which prevents the interaction between VCA regions with Arp2/3 complex[11]. Tomasevic et al demonstrated that PIP2 negatively regulates WASP, but not N-WASP activation mediated by Cdc4215
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