The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1.
We observed evolutionary conservation of canonical nuclear localization signal sequences (K(K/R)X(K/R)) in the C-terminal polybasic regions (PBRs) of some Rac and Rho isoforms. Canonical D-box sequences (RXXL), which target proteins for proteasome-mediated degradation, are also evolutionarily conserved near the PBRs of these small GTPases. We show that the Rac1 PBR (PVKKRKRK) promotes Rac1 nuclear accumulation, whereas the RhoA PBR (RRGKKKSG) keeps RhoA in the cytoplasm. A mutant Rac1 protein named Rac1 (pbrRhoA), in which the RhoA PBR replaces the Rac1 PBR, has greater cytoplasmic localization, enhanced resistance to proteasome-mediated degradation, and higher protein levels than Rac1. Mutating the D-box by substituting alanines at amino acids 174 and 177 significantly increases the protein levels of Rac1 but not Rac1(pbrRhoA). These results suggest that Rac1 (pbrRhoA) is more resistant than Rac1 to proteasome-mediated degradative pathways involving the D-box. The cytoplasmic localization of Rac1(pbrRhoA) provides the most obvious reason for its resistance to proteasome-mediated degradation, because we show that Rac1(pbrRhoA) does not greatly differ from Rac1 in its ability to stimulate membrane ruffling or to interact with SmgGDS and IQGAP1-calmodulin complexes. These findings support the model that nuclear localization signal sequences in the PBR direct Rac1 to the nucleus, where Rac1 participates in signaling pathways that ultimately target it for degradation.
Molecular Pharmacology Laboratory, Guthrie Research Institute, Sayre, Pennsylvania 18840, USA.
Cathy Cole
CC
Lanning
Janelle L
JL
Daddona
Rebecca
R
Ruiz-Velasco
Shulamith H
SH
Shafer
Carol L
CL
Williams
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000409:Alanine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde