ABSTRACT
High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1-DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2â Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.
Subject(s)
DNA/metabolism , HMG-Box Domains , HMGB1 Protein/chemistry , HMGB1 Protein/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , DNA/chemistry , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Rats , Sequence AlignmentABSTRACT
Rho family GTPases control a diverse range of cellular processes, and their deregulation has been implicated in human cancer. Guanine nucleotide dissociation inhibitors (GDIs) bind and sequester GTPases in the cytosol, restricting their actions. RhoGDI2 is a member of the GDI family that acts as a metastasis suppressor in a variety of cancer types; however, very little is known about the regulation of this protein. Here, we present a mechanism for inactivation of RhoGDI2 via protein kinase C (PKC) phosphorylation of Ser31 in a region that contacts GTPases. In cells, RhoGDI2 becomes rapidly phosphorylated at Ser31 in response to phorbol 12-myristate 13-acetate stimulation. Based on the effects of pharmacological inhibitors and knockdown by siRNA, we determine that conventional type PKCα is responsible for this phosphorylation. Phospho-mimetic S31E-RhoGDI2 exhibits reduced binding to Rac1 relative to wild type, with a concomitant failure to reduce levels of activated endogenous Rac1 or remove Rac1 from membranes. These results reveal a mechanism of downregulation of RhoGDI2 activity through PKC-mediated phosphorylation of Ser31. We hypothesize that this mechanism may serve to neutralize RhoGDI2 function in tumors that express RhoGDI2 and active PKCα.
