Nuclear translocation of CBP/p300-interacting protein CITED1 induced by parathyroid hormone requires serine phosphorylation at position 79 in its 63–84 domain

Abstract

The transcriptional cofactor CITED1 inhibits osteoblastic differentiation and blunts the stimulation of osteoblastic differentiation by parathyroid hormone (PTH). In the MC3T3-E1 osteoblastic cell line, we found that CITED1 was located predominantly in the cytoplasm and that hPTH(1–34) increased translocation of CITED1 from the cytoplasm to the nucleus. This response to hPTH(1–34) was not observed when all 9 serine residues within the 63–84 domain of CITED1 were mutated to alanines (CITED1 9S>A) or when a single serine to alanine mutation was made at position 79 (CITED1 S79>A). CITED1 containing mutations of these 9 serines to glutamic acid (9S>E) retained the same nuclear translocation response to hPTH(1–34) as the wild type CITED1. ALP activity and formation of mineralized nodules were inhibited in cells transfected with pcDNA3-CFP-CITED1 or with pcDNA3-CFP-CITED1 9S>E with or without hPTH(1–34) treatment (all P<0.05); these changes were not observed using CITED1 9S>A. Cells exposed to intermittent treatment with hPTH(1–34) expressed more ALP2, Runx2 and osteocalcin than vehicle-treated cells. These effects of hPTH(1–34) were inhibited in cells transfected with pcDNA3-CFP-CITED1 or pcDNA3-CFP-CITED1 9S>E, but were slightly enhanced by the alanine mutants. PKC activator (TPA) increased nuclear translocation of CITED1, whereas a PKC inhibitor (Go6983) blunted the effect of hPTH(1–34) on the nuclear translocation of wildtype CITED1 but not of CITED1 S79>E. The data indicated that serine phosphorylation at position 79 in the 63–84 domain is associated with PKC activation, and is required for both CITED1 nuclear translocation induced by PTH and the negative effects of CITED1 on osteoblastic differentiation and mineralization.