Fig. 3: CRL3-KLHL4 interacts with the GIT-PIX-PAK signaling module through GIT1 and utilizes GIT-PIX complexes as co-adaptors to multi-monoubiquitylate PAK1. | Nature Communications

Fig. 3: CRL3-KLHL4 interacts with the GIT-PIX-PAK signaling module through GIT1 and utilizes GIT-PIX complexes as co-adaptors to multi-monoubiquitylate PAK1.

From: A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development

Fig. 3: CRL3-KLHL4 interacts with the GIT-PIX-PAK signaling module through GIT1 and utilizes GIT-PIX complexes as co-adaptors to multi-monoubiquitylate PAK1.The alternative text for this image may have been generated using AI.

a CRL3-KLHL4 engages the GIT-PIX-PAK signaling module through GIT1, as evidenced by immunoblot analysis of anti-FLAG IPs from control or GIT1-depleted HEK293T cells. n = 2 biological replicates. b Three positively charged residues in GIT1 are essential for KLHL4 binding. Immunoblot analysis of anti-FLAG IPs from HEK293T cells transfected with indicated constructs. n = 2 biological replicates. GIT1 = protein isoform 3 on UniProt. c KLHL4 possesses three acidic patches on the bottom of its KLECH propeller. Depicted is a surface charge map of a structural model of KLHL4. The upper panel shows the top of the KELCH β-propeller, the canonical binding site for substrates in other BTB-KELCH proteins (indicated with black dotted circle), which exhibits a positively charged groove. The lower panel depicts the bottom of the KELCH β-propeller that contains three acidic patches. d Acidic patch 1 (ΔGIT1), but not acidic patch 2 or 3 (AP2 and AP3), in KLHL4 mediates binding to the GIT-PIX-PAK signaling module. Immunoblot analysis of anti-FLAG IPs from HEK293T cells transfected with indicated constructs. n = 3 biological replicates. e GIT-PIX complexes recruit PAK1 to KLHL4. GIT1-depleted HEK293T cells were co-transfected with indicated constructs, lysed, and subjected to anti-FLAG-IP followed by immunoblotting with denoted antibodies. KLHL4 can only bind PAK1 when shRNA-resistant GIT1 is expressed, and this association can be blocked by co-expression of PAK-binding-deficient PIX proteins (α/β-PIX SH3*). n = 1 biological replicate. f CRL3-KLHL4 ubiquitylates PAK1 in cells. Immunoblot analysis of denaturing HISubiquitin pulldowns from RPE-1 cells expressing denoted combinations of constructs using the indicated antibodies. PCNA = control for general ubiquitylation efficiency. n = 3 biological replicates. g PAK1 requires interactions with GIT-PIX complexes to be ubiquitylated by CRL3-KLHL4 in cells. Immunoblot analysis of denaturing HISubiquitin pulldowns from RPE-1 cells expressing denoted combinations of constructs. ΔPIX = GIT-PIX-complex-binding-deficient PAK1. Ubiquitin = control for general ubiquitylation efficiency. n = 2 biological replicates. h Model how CRL3-KLHL4 uses GIT1-PIX complexes as co-adaptors to bind and ubiquitylate PAK1.

Back to article page