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  • Previous studies have indicated that hSSB may be phosphoryla

    2020-09-16

    Previous studies have indicated that hSSB1 may be phosphorylated at threonine residue 117 (T117) [13] and acetylated at lysine residue 94 (K94) [15], both of which promote hSSB1 function in response to ionising radiation exposure. Mass spectrometry screens of proteins immunoprecipitated with phosphorylation and acetylation motif specific Nemadectin mg have also identified numerous other potentially modified hSSB1 residues, although these remain to be validated and have not been associated with any particular stress or cellular function [16]. Nevertheless, these findings support that hSSB1 may be highly regulated in cells through post-translational modifications.
    Materials and methods
    Results
    Discussion Enzymatically active DNA-PK is a central component of the cellular response to replication fork inhibition [6]. For instance, cells depleted of endogenous DNA-PKcs or expressing an enzymatically deficient kinase mutant are unable to efficiently restart stalled replication forks and show reduced clonogenic survival when treated with hydroxyurea [7]. With the notable exception of RPA32 [8], as well as of auto-phosphorylation [7], few relevant DNA-PK substrates have however been identified in response to replication stress. In this article we demonstrate that hSSB1 S134 phosphorylation is required for clonogenic survival of cells treated with the replication stress compounds hydroxyurea, aphidicolin and camptothecin, as well as establish that phosphorylation is primarily a result of DNA-PK activity. As a small decrease in hSSB1 S134 phosphorylation was also observed following the inhibition of ATM and ATR, we cannot however exclude that these kinases may also contribute a small amount to the phosphorylation of hSSB1 S134. Such overlap is consistent with previous studies on other known PI3K-like kinase substrates and is exemplified by the overlapping ATM and DNA-PK phosphorylation of RPA32 residues S4/8 and S12 [8], as well as the ATM and DNA-PK phosphorylation of Chk1 S317 in cells where ATR (the predominant kinase of Chk1 S317) function is suppressed (25). Alternatively, the reduction in hSSB1 S134 phosphorylation observed following ATM and ATR inhibition might result from indirect de-regulation of DNA-PK function. Indeed, the phosphorylation and thereby activation of DNA-PK by ATM [31] and ATR [32] has previously been demonstrated, the disruption of which might also explain the small reduction in hSSB1 S134 phosphorylation observed following inhibition of these enzymes. While hSSB1 S134 is also phosphorylated by DNA-PK in undamaged cells, this may be maintained at steady-state levels by the opposing activities of PPP-family protein phosphatases. This family includes numerous members that are inhibited by okadaic acid in cells, including PP2A, PP4, PP5, and PP6 (23). The increase in hSSB1 phosphorylation observed following treatment with okadaic acid therefore suggests the involvement of one of these enzymes in the de-phosphorylation of hSSB1. Indeed, each of these enzymes has previously been implicated in the regulation of DNA repair pathways [33] and may represent plausible regulators of hSSB1. Although we did observe an additional increase in hSSB1 phosphorylation when calcyculin A was added to cells, a compound that also inhibits PP1, it is unclear whether the greater affect observed here with comparison to okadaic acid treatment is due to PP1 involvement. Indeed, calyculin A also shows increased potency towards PP5, the involvement of which also represents a plausible explanation. Further studies will therefore be required to establish which PPP-family members are involved in dephosphorylating hSSB1 S134. Such experiments, which would likely require the specific depletion of individual PPP-family member subunits, may however be hampered by potential indirect affects which may arise due to disruption of dephosphorylation events required for proper regulation of DNA-PK or of DNA-PK regulatory proteins. Indeed, we cannot exclude the possibility that such indirect affects may account for some of the increased hSSB1 S134 phosphorylation observed here following calyculin A and okadaic acid treatment. In addition, as both of these compounds are know to have cytotoxic affects on cells [34], we cannot exclude, based on our data, that hSSB1 S134 may be phosphorylated as a result of these cellular changes, independently of direct disruption of dynamic phosphorylation.