Krit1 contains a PTB domain that acts as a passport for nuclear translocation

Mutations in the KRIT1 gene have been shown to cause Cerebral Cavernous Malformations, which are vascular malformations mostly located in the central nervous system, occurring in 0.1 to 0.5% of the population and predisposing to seizures, intracerebral haemorrhage and focal neurological deficits. The KRIT1 gene encodes for a 736-amino acid protein whose cellular functions are not yet defined. However, it is known that the N-terminal portion of KRIT1 contains an NPXY motif that constitutes the binding site for ICAP1A (integrin cytoplasmic domain-associated protein-1A), a PTB domain-containing protein that we have recently characterized as a nucleocytoplasmic shuttling protein able to modulate both beta1 integrin adhesive functions and gene expression. In addition, the central region of KRIT1 contain three putative ankyrin repeats, which are known to mediate protein-protein interactions, whereas the C-terminal portion contains a FERM domain and a putative binding site for RAP1A (, a GTPase involved in integrin and cadherin signaling, suggesting that KRIT1 might act as a scaffold protein for the assembly of molecular complexes involved in the regulation of cell adhesion-related processes. Recently, we have reported the identification of KRIT1B, a novel KRIT1 isoform characterized by the alternative splicing of the 15th coding exon. Here we report that KRIT1B localizes exclusively in the cytoplasm and is devoid of a PTB-like subdomain contained in the lobe F3 of the FERM domain. On the contrary, we demonstrate that the canonical form KRIT1A undergoes a dynamic shuttling between the cytoplasm and the nucleus which relies on either the PTB subdomain of the FERM domain or the N-terminal NPXY motif. Indeed, using bioinformatic analyses we show that an intramolecular self-association may occur between the C-terminal PTB subdomain and the N-terminal NPXY motif of KRIT1A, suggesting a conformationally regulated mechanism for the nucleocytoplasmic shuttling. In addition, we show that KRIT1B is not able to interact with RAP1A, while its expression consistently prevents the nuclear localization of ICAP1A. Our data define a mechanism for the nucleocytoplasmic shuttling of KRIT1A and suggest that the KRIT1B isoform may act as a dominant negative regulator of ICAP1A nuclear functions, thus opening new ways for understanding the role of KRIT1 in CCM pathogenesis.