泛素化是蛋白质稳定性和功能的关键调节剂。已知多功能蛋白p27在K48连接的泛素化后被蛋白酶体降解。然而,我们最近报道,当泛素结合酶UbcH7(UBE2L3)过表达时,p27稳定,细胞周期在多种不同的细胞类型,包括眼晶状体,视网膜,HEK-293和HELA细胞。但是与p27的这种稳定相关的泛素连接酶仍然是个谜。从体外泛素化筛选开始,我们确定RSP5为酵母E3连接酶伴侣的UbcH7在p27的泛素化。对E3连接酶的同源人NEDD4家族的筛选表明,SMURF1而不是其紧密的同源物SMURF2可以稳定细胞中的p27。我们发现SMURF1在体外用K29O泛素化p27而不是K29R或K63O泛素。表现出对K29链形成的强烈偏好。与SMURF1/UbcH7对p27的稳定一致,我们还发现SMURF1,UbcH7和p27促进细胞迁移,而SMURF1或UbcH7的敲减会降低细胞迁移。我们进一步证明了SMURF1/p27和UbcH7/p27在迁移细胞前缘的共定位。总之,这些结果表明,SMURF1和UbcH7共同在p27上产生K29连接的泛素链,从而稳定p27并促进其细胞周期无关的调节细胞迁移的功能。
Ubiquitination is a key regulator of protein stability and function. The multifunctional protein
p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of
p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes
p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of
p27, we also found that SMURF1, UbcH7, and
p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/
p27 and UbcH7/
p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.