Aurothiomalate Inhibits Transformed Growth by Targeting the PB1 Domain of Protein Kinase Cι

Abstract

We recently identified the gold compound aurothiomalate (ATM) as a potent inhibitor of the Phox and Bem1p (PB1)-PB1 domain interaction between protein kinase C (PKC) and the adaptor molecule Par6. ATM also blocks oncogenic PKC signaling and the transformed growth of human lung cancer cells. Here we demonstrate that ATM is a highly selective inhibitor of PB1-PB1 domain interactions between PKC and the two adaptors Par6 and p62. ATM has no appreciable inhibitory effect on other PB1-PB1 domain interactions, including p62-p62, p62-NBR1, and MEKK3-MEK5 interactions. ATM can form thio-gold adducts with cysteine residues on target proteins. Interestingly, PKC (and PKC) contains a unique cysteine residue, Cys-69, within its PB1 domain that is not present in other PB1 domain containing proteins. Cys-69 resides within the OPR, PC, and AID motif of PKC at the binding interface between PKC and Par6 where it interacts with Arg-28 on Par6. Molecular modeling predicts formation of a cysteinyl-aurothiomalate adduct at Cys-69 that protrudes into the binding cleft normally occupied by Par6, providing a plausible structural explanation for ATM inhibition. Mutation of Cys-69 of PKC to isoleucine or valine, residues frequently found at this position in other PB1 domains, has little or no effect on the affinity of PKC for Par6 but confers resistance to ATM-mediated inhibition of Par6 binding. Expression of the PKC C69I mutant in human non-small cell lung cancer cells confers resistance to the inhibitory effects of ATM on transformed growth. We conclude that ATM inhibits cellular transformation by selectively targeting Cys-69 within the PB1 domain of PKC.

Matrix metalloproteinase-10 is a critical effector of protein kinase Cι-Par6α-mediated lung cancer

“…Likewise ATM, which specifically binds the PB1 domain of PKCi, inhibits anchorage-independent growth of NSCLC cells (Erdogan et al, 2006;Stallings-Mann et al, 2006). Therefore, we assessed the ability of wild-type PKCi and two PB1 domain mutants of PKCi (PKCi-K20A and PKCi-D63A) to support anchorage-independent growth in PKCi-RNAi cells (Figure 1a).…”

“…The gold salt aurothiomalate (ATM) selectively inhibits the PB1-PB1 domain interaction between PKCi and Par6 in vitro by binding to cysteine 69 within the PB1 domain of PKCi (Erdogan et al, 2006;Stallings-Mann et al, 2006). ATM blocks Rac1 activity and inhibits anchorage-independent growth of NSCLC cells, suggesting a role for PB1-PB1 domain interactions between PKCi and Par6 in Rac1 activation and transformation (Erdogan et al, 2006;Stallings-Mann et al, 2006).…”

Structural and biochemical insights into the homotypic PB1-PB1 complex between PKCζ and p62

“…Remarkably, mutating Cys68 to residues with bulky side chains (Tyr, Trp, Phe or Arg) led to complete loss of the PKCζ-p62 interaction, whereas substitution by Val (or Ile, data not shown), the corresponding residue(s) in other PB1 domains, had little effect. These data, together with previous results [38], indicate that the conserved cysteine within the OPCA motif of aPKC-PB1 domains is the determinant for ATM inhibition of the interactions between aPKCs and other PB1-containing proteins with a functional basic surface.…”

“…On the contrary, the ATM-modified PKCζ-PB1 failed to bind to p62-PB1 ( Figure 6A), suggesting that ATM modifies PKCζ-PB1 and impedes its interaction with p62-PB1. ATM was suggested to selectively target a unique cysteine residue, Cys69, within the OPCA motif of PKCι-PB1 [38]. However, there are four cysteine residues in both aPKC-PB1 sequences, and these thiol groups are all exposed to solvent in their structures ( Figures 1B and 3A).…”

“…These compounds have been shown to effectively modify PKCι and ablate its association with Par-6 [38][39][40]. Considering that PKCζ and PKCι are highly conserved in both sequence and structure, we examined the effect of ATM modification on the interaction between PKCζ and p62.…”