Phosphorylation and Alternative Splicing of 7B2 Reduce Prohormone Convertase 2 Activation

Mol Endocrinol. 2015 May;29(5):756-64. doi: 10.1210/me.2014-1394. Epub 2015 Mar 26.

Abstract

FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alternative Splicing
  • Amino Acid Sequence
  • Casein Kinase I / metabolism
  • Endoplasmic Reticulum / metabolism
  • Enzyme Activation
  • Extracellular Matrix Proteins / metabolism
  • HEK293 Cells
  • Heat-Shock Proteins / metabolism
  • Humans
  • Molecular Sequence Data
  • Neuroendocrine Secretory Protein 7B2 / genetics
  • Neuroendocrine Secretory Protein 7B2 / metabolism*
  • Phosphorylation
  • Proprotein Convertase 2 / metabolism*
  • Protein Processing, Post-Translational

Substances

  • Extracellular Matrix Proteins
  • Heat-Shock Proteins
  • Neuroendocrine Secretory Protein 7B2
  • SCG5 protein, human
  • Casein Kinase I
  • FAM20C protein, human
  • Proprotein Convertase 2
  • molecular chaperone GRP78