KCTD13基因敲除HEK293细胞

The potassium channel tetramerization domain containing 13 (KCTD13) protein is a substrate-specific adapter for cullin3-based E3 ubiquitin ligase. Patients with copy number variants at this locus exhibit genitourinary tract anomalies. In this study, we show that decreased androgen receptor (AR) protein level correlated with increased AR ubiquitination in the testis of Kctd13-deficient mice, suggesting that KCTD13 inhibits AR ubiquitination. KCTD13 increased CUL3-dependent AR ubiquitination but had no effect on CUL3 binding to AR, confirming the role of KCTD13 as an adaptor of CUL3 ligase. Recombinant KCTD13 directly binds to recombinant AR, and the BTB domain of KCTD13 is critical for binding both the N-terminal domain of AR and STUB1. Moreover, KCTD13 dose-dependently decreased STUB1 binding to AR resulting in decreased AR ubiquitination. KCTD13 ΔBTB was unable to bind to AR and subsequently failed to block STUB1-mediated AR ubiquitination, strongly suggesting that reduced AR ubiquitination is dependent on KCTD13 ability to dissociate AR/STUB1 complex. Furthermore, KCTD13 increased the expression of AR target gene, FOXJ1, whereas KCTD13 ΔBTB had no effect. Our data reveal a distinctive mode of action of KCTD13 on AR ubiquitination depending on the E3 ubiquitin ligase involved: (1) KCTD13 increased CUL3-dependent AR ubiquitination but had no effect on CUL3 binding to AR; and (2) KCTD13 decreased STUB1-mediated AR ubiquitination by decreasing STUB1 binding to AR thus preventing AR ubiquitination. We hypothesize that in the testes of Kctd13-deficient mice, the absence of KCTD13 results in increased binding of STUB1 to AR leading to increased AR ubiquitination and degradation.

Precise control of neuronal migration is required for the laminar organization of the neocortex and critical for brain function. We previously reported that the acute disruption of the gene ( conditional knock-out; cKO) during mouse embryogenesis causes anomalous neuronal migration in the neocortex, but paradoxically the cKO did not have a cortical phenotype, suggesting some forms of compensation exist. In this study, we report that MST3, another member of the GCKIII subgroup of the Ste20-like kinase family, compensates for loss of and vice versa with sex independent manner. MST3 overexpression rescued neuronal migration deficit and abnormal axonogenesis in cKO brains. Mechanistically, STK25 leads to Rac1 activation and reduced RhoA levels in the developing brain, both of which are required to fully restore neuronal migration in the cKO brain. Abnormal migration phenotypes are also rescued by overexpression of Bacurd1and Cul3, which target RhoA for degradation, and activate Rac1. This study reveals that MST3 upregulation is capable of rescuing acute deficiency and resolves details of signaling downstream STK25 required for corticogenesis both common to and distinct from MST3 signaling. Proper neuronal migration during cortical development is required for normal neuronal function. Here, we show that STK25 and MST3 kinases regulate neuronal migration and polarization in a mutually compensatory manner. Furthermore, STK25 balances Rac1 activity and RhoA level through forming complexes with α-PIX and β-PIX, GTPase regulatory enzymes, and Cullin3-Bacurd1/Kctd13, a pair of RhoA ubiquitination molecules in a kinase activity-independent manner. Our findings demonstrate the importance of overlapping and unique roles of STK25 and MST3 to regulate Rho GTPase activities in cortical development.

OBJECTIVE:Micropenis is a condition with significant physical and psychological implications caused mainly by decreased androgen action in penile development. Kctd13-knockout (Kctd13-KO) mice have micropenis, cryptorchidism, and fertility defects because of reduced levels of androgen receptor (AR) and SOX9. We hypothesized that normalizing the levels of AR and SOX9 in the Kctd13-KO penis could help us to understand the mechanism of action of these signaling pathways on penile development. METHODS:We generated transgenic mice lacking Kctd13 and conditionally expressing AR in the urethral mesenchyme after Cre activation with Twist2 (Kctd13-KO; AR-CMV; Twist2; herein called AR+), and Sox9 in the urethral epithelium after Cre activation with Shh (Kctd13-KO; Sox9-CAG; Shh; herein called SOX9+). Mice penile morphology, fertility, and the effect of KCTD13 on AR and SOX9 ubiquitination were evaluated. RESULTS AND DISCUSSION:Kctd13-KO micropenis phenotype was rescued after increasing levels of penile AR or SOX9 as transgenic AR+ and SOX9+ mice have longer penile lengths than Kctd13-KO mice and are comparable to WT mice. In addition, male-urogenital-mating-protuberance and the baculum were significantly shorter and narrower in Kctd13-KO mice compared with transgenic AR+ and SOX9+ mice. The position of the urethral meatus was similar and orthotopic in location in Kctd13-KO, AR+, SOX9+, and WT penises indicating that none of these mice had hypospadias. The subfertility of AR+ and SOX9+ mice was improved. The ectopic expression of KCTD13 in HEK293 cells strongly reduced AR ubiquitination which is abolished when the proteasome pathway is inhibited and this process is mediated by the ubiquitin ligase, STUB1. The effect of KCTD13 on SOX9 ubiquitination is minimal. CONCLUSION:KCTD13 regulates AR ubiquitination by modulating STUB1 binding to AR. Penile restoration of AR and SOX9 improved penile development in Kctd13-KO mice allowing us to discern the contribution from individual signaling pathways and cell types in penile development.