Poly-ADP-ribosylation reactions occur both in multi- and unicellular organisms and play a major role in a wide range of biological processes, such as maintenance of genomic stability, transcriptional regulation and cell death (reviewed in [1, 2]). The enzyme responsible for the synthesis of poly-ADP-ribose was named poly-ADP-ribose polymerase (PARP) (reviewed in [1, 2]). For a long time, PARP-1 was thought to be the only enzyme with poly-ADP-ribosylation activity in mammalian cells; however, primary cells derived from parp-1 knockout mice can still synthesize poly-ADP-ribose polymers after DNA damage . This led to the identification of five novel poly-ADP-ribosylating enzymes, indicating that PARP-1 belongs to a family of at least six members ([4–6] and reviewed in [1, 2]). PARP-2 and PARP-1 can homo- and heterodimerize and display partially redundant functions as indicated by the embryonic lethality of the parp1-parp2-double gene disruption ( and reviewed in ).
Mouse PARP-2 was described as a 66 kDa nuclear protein with poly-ADP-ribosylating activity . The amino-terminal region of PARP-2 (aa 1–90), containing the DNA binding SAP domain, has no significant homology with any other PARP . However, it is rich in basic amino acids (27% Lys or Arg), which are likely to be involved in DNA binding (reviewed in ). On the other hand, these basic residues could be involved in the nuclear and/or nucleolar targeting of the protein . Previous studies suggested that the nuclear localization signal (NLS) of mPARP-2 is indeed located in the amino-terminal part between aa 1–69 of the protein [9, 11]. Meder et al. postulated a bipartite NLS for PARP-2, but did not provide further experimental evidences to support their hypothesis . Interestingly, the amino-terminal region of human and mouse PARP-2 shows higher sequence variability compared to the highly conserved carboxy-terminal catalytic region (62% identity between the amino-terminus of mPARP-2 and hPARP-2). In cells, PARP-2 has been described to regulate different processes via protein-protein interactions mediated by its amino-terminal domain (aa 1–208; reviewed in ).
Karyopherins, including both importins and exportins, consitute a conserved family of mobile targeting receptors that mediate the bidirectional trafficking of macromolecules across the nuclear envelope [12, 13]. Most karyopherins interact directly with cargo molecules that contain nuclear import and export signals. However, importin α functions as an adaptor that links classical NLS (cNLS)-containing proteins to importin β, which, in turn, docks the ternary complex at the nuclear-pore complex (NPC). The importin α/β heterodimer is predicted to target hundreds of proteins to the NPC and facilitate their translocation across the nuclear envelope . The importin α gene family has undergone considerable expansion during the course of eukaryotic evolution. Whereas the yeast S. cerevisiae genome encodes a single importin α, the human genome encodes six genes that fall into three phylogenetically distinct groups .
The nuclear targeting signal in the simian virus 40 (SV40) large T antigen was characterized more than 20 years ago [16, 17]. Since then, several pathways for nucleocytoplasmic transport have been described, of which the classical nuclear import pathway is the best characterized. cNLSs are typified by either a single cluster of basic amino acids (monopartite NLS) or two clusters of basic amino acids separated by a 10–12 amino acid linker (bipartite NLS). The SV40 large T antigen (PKKKRKV) and nucleoplasmin (KRPAATKKAGQAKKKK) cNLSs are the prototypic monopartite and bipartite cNLS [18, 19]. Through alanine scanning of the Myc, monopartite SV40, and artificial bipartite SV40 cNLS, Hodel and colleagues found that the binding affinity of a cNLS for importin measured in vitro correlated with the steady state nuclear accumulation and import rate of the corresponding cNLS cargo in vivo [20, 21].
Here, we demonstrate that lysine 36 in the DNA binding domain (DBD) of PARP-2, which lies within a predicted cNLS motif, is required for complex formation with the importin proteins and subsequent nuclear import of PARP-2.