Direct regulation of Chk1 protein stability by E3 ubiquitin ligase HUWE1

Abstract

The HECT E3 ubiquitin ligase HUWE1 is required for a wide array of important functions in cell biology. Although HUWE1 is known to play a role in DNA damage signaling, the mechanism(s) that underlie this function remain elusive. HUWE1 regulates effectors of DNA replication and genotoxic stress tolerance. However, the loss of HUWE1 can also result in the accrual of significant endogenous DNA damage due to insufficient remediation of replication stress induced by an overabundance of key substrates. We discovered that HUWE1 depletion leads to a significant increase in levels of the single-strand break effector kinase Chk1, independent of the DNA damage response, activation of apical DNA damage repair (DDR) signaling kinases (ATM and ATR), and the tumor suppressor p53. We also identified multiple lysine residues on Chk1 that are polyubiquitinated by HUWE1 in vitro, many of which are within the kinase domain. HUWE1 knockdown also markedly prolonged the protein half-life of Chk1 in steadystate conditions and resulted in greater stabilization of Chk1 protein than depletion of Cul4A, an E3 ubiquitin ligase previously described to control Chk1 abundance. Moreover, prolonged replication stress induced by hydroxyurea or camptothecin resulted in a reduction of Chk1 protein levels, which was rescued by HUWE1 knockdown. Our study indicates that HUWE1 plays a significant role in the regulation of the DDR signaling pathway by directly modulating the abundance of Chk1 protein.

Beyond K48 and K63: non-canonical protein ubiquitination

“…The DNA damage response (DDR) has been repeatedly shown to be controlled by K6-linked ubiquitination [ 21 – 25 ]. Pioneering studies reported K6-linked auto-ubiquitination of BRCA1-BARD1, a major DDR complex [ 21 ], and demonstrated the formation of K6-linked chains during replication stress and double-strand break repair [ 22 , 26 ].…”

HUWE1 mediates inflammasome activation and promotes host defense against bacterial infection

“…tumorigenesis (50)(51)(52)(53)(54). HUWE1 also plays important roles in regulating the DNA damage response (DDR) by targeting histones, MCL-1, and Chk1 (44,45,55,56). In addition, clinical evidence and exome sequencing revealed that HUWE1 mutations cause X chromosome-linked intellectual disability (57)(58)(59).…”

Modular HUWE1 architecture serves as hub for degradation of cell-fate decision factors

“…The HECT family member HUWE1 (HECT, UBA and WWE domain containing protein 1, also known as Mule/ARF-BP1/LASU1/HECTH9) was initially shown to degrade three key regulators of stress and survival: Mcl1, p53, and Myc [6][7][8] . A growing list of HUWE1 substrates has since been reported 9 , including the stress-responsive regulator of mTORC1 signaling, DDIT4 10,11 , and many DNA damage response factors, such as the BRCA1 tumor suppressor, TopBP1, Cdc6 and CHEK1 [12][13][14][15] . Loss of HUWE1 sensitizes cells not only to DNA damage, but also to a variety of other stressors, including both oxidative and hypoxic stress 9,[16][17][18][19] .…”