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Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells
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Structure-Function Analysis of p57KIP2 in the Human Pancreatic Beta Cell Reveals a Bipartite Nuclear Localization

Lauryn Choleva1, Peng Wang2, Hongtao Liu2

  • 1Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Endocrinology
|December 28, 2023
PubMed

Insights

Mutations in CDKN1C cause pediatric endocrine syndromes by affecting p57KIP2, a cell cycle inhibitor. This study reveals p57KIP2 has two nuclear localization sequences essential for its function in human beta cells.

Area of Science:

  • Endocrinology
  • Molecular Biology
  • Genetics

Background:

  • Mutations in CDKN1C, encoding the cell cycle inhibitor p57KIP2, are linked to pediatric endocrine syndromes.
  • The precise structure and function of p57KIP2 within human pancreatic beta cells remain largely uncharacterized.
  • p57KIP2 is primarily localized to the nucleus in human beta cells, suggesting a role for its nuclear localization sequence (NLS) in disease pathogenesis.

Purpose of the Study:

  • To conduct a comprehensive structure-function analysis of CDKN1C/p57KIP2.
  • To investigate the role of p57KIP2's nuclear localization in human beta cell function and disease.
  • To identify and characterize the nuclear localization sequences (NLS) of p57KIP2.

Main Methods:

  • Utilized RIP1 insulin promoter-driven adenoviruses to express p57KIP2 deletion constructs in beta cells.
  • Employed real-time polymerase chain reaction and immunoblotting to confirm p57KIP2 expression and specificity.
  • Applied immunocytochemistry to determine the subcellular localization of wild-type and mutant p57KIP2.

Main Results:

  • Wild-type p57KIP2 demonstrated nuclear localization in human beta cells.
  • Deletion of a putative NLS (amino acids 278-281) prevented nuclear entry.
  • A second NLS (amino acids 312-316) was identified, with both NLS regions being necessary, but not sufficient, for nuclear localization.

Conclusions:

  • p57KIP2 possesses a bipartite NLS comprising two distinct clusters of positively charged amino acids.
  • Variants affecting these NLS sequences lead to loss of functional p57KIP2 and subsequent beta cell expansion in human disease.
  • Understanding p57KIP2 NLS function is crucial for deciphering the molecular mechanisms underlying pediatric endocrine syndromes associated with CDKN1C mutations.
Keywords:
Beckwith-Wiedemann syndromebeta celldiabetes

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