Benjamin E. Turk

The human genome encodes more than 60 proteins containing Rho GTPase activating protein (RhoGAP) domains, many of which remain understudied with respect to their target specificity and biological roles. SYDE1 and SYDE2 are two such orphan RhoGAPs, for which there are few studies characterizing their biochemical and cellular functions and conflicting reports identifying their cognate GTPases. We previously identified SYDE1 and SYDE2 in a screen for substrates of the c-Jun N-terminal kinases. Here, we show that SYDE1 and SYDE2 are preferentially phosphorylated by JNK1 relative to other mitogen-activated protein kinases (MAPKs) at sites proximal to a kinase docking region. Purified SYDE1 and SYDE2 are shown to have significant catalytic GAP activity toward RhoA, Rac1, and Cdc42. However, neither up-nor down-regulation of SYDE1/2 expression leads to detectable changes in bulk GTP loading of any of these GTPases. Nevertheless, we demonstrate that SYDE1 and SYDE2, in a partially GAP-dependent manner, increase cell spreading and number of focal adhesions, and promote more directionally persistent migration in HEK293 cells. Together, these findings establish SYDE1 and SYDE2 as robust JNK substrates with catalytic activity toward a set of Rho GTPases and reveal basic functions of SYDE1 and SYDE2 in regulating cell morphology, adhesion, and migration. One sentence summary: A study of RhoGAPs SYDE1 and SYDE2 reveals their core biochemical and functional characteristics, and establishes their importance in cell morphology, adhesion, and migration.

Differential expression of SKO vs YRG cells ± IFNAR blockade by RNAseq.

Summary of exonic mutations in YRG cells compared with YMG and WT C57BL6/J cells.

Differential expression of SKO vs YRG cells, by RNAseq.

Figure S2: CRISPR screen guide distributions, related to Figure 1

Figure S1: Experimental validation of SETDB1 knockout and flow gating schemes, related to Figure 4

Figure S6: Characterization of possible SKO tumor antigens, related to Figure 4.

List of primers used to perform RT-qPCR on YUDOSO cells related to Figure S6.

Figure S5: Evaluation of SETDB1 knockdown in human melanoma cells, related to Figure 4:

Figure S4: Evaluation of differences in SKO tumor gene expression and microenvironment profiles, related to Figure 3: