Structure-Function Analysis of p57KIP2 in the Human Pancreatic Beta Cell Reveals a Canonical Bipartite Nuclear Localization Sequence (NLS)

p57KIP2 is a canonical cell cycle inhibitor encoded by the CDKN1C gene on chromosome 11. Mutations in CDKN1C underlie multiple pediatric endocrine syndromes including Beckwith-Wiedemann Syndrome (BWS), the Focal Variant of Congenital Hyperinsulinism (FoCHI), Insulinoma, Russel-Silver Syndrome and IMAGe Syndrome. Despite this central role in disease, little is known about the function of p57KIP2 in any cell type, including the human pancreatic beta cell. We hypothesized that p57KIP2 has functions that extend beyond cell cycle control and are unique to the human beta cell. We prepared RIP1 insulin promoter-driven adenoviruses encoding deletions of multiple putatively important and disease-associated but unexplored regions of p57KIP2 (WTp57, p57ΔPAPA, p57ΔCDKI, p57ΔPCNA and p57ΔNLS), and performed the first comprehensive structure-function analysis of CDKN1C/p57KIP2 in the human beta cell. RT-PCR and immunoblot analyses confirmed p57KIP2 overexpression, construct size and beta cell specificity in human islets. By immunocytochemistry, all constructs demonstrated nuclear localization, with the exception of a deletion mutant removing a putative NLS at amino acids 278-281. Unexpectedly, we identified a second NLS at amino acids 312-316. Further analysis showed that each individual NLS is required for nuclear localization of p57KIP2, but neither alone is sufficient to permit nuclear localization of p57KIP2. Finally, constructs containing both the 278-281 and 312-316 sequences were necessary and sufficient to target heterologous proteins to the nucleus of human beta cells. In summary, we discovered that p57KIP2 contains a classic bipartite NLS characterized by two clusters of positively charged amino acids 278KRKRS281 and 312RKRLR316 separated by a proline-rich linker region. Variants in the sequences encoding these two NLS sequences account for functional p57KIP2 loss and beta cell expansion seen in human disease. Disclosure L. Choleva: None. P. Wang: None. H. Liu: None. O. Wood: None. L. Lambertini: None. E. Karakose: None. A.F. Stewart: None.