The
14-3-3 protein family consists of acidic 30-kDa
proteins composed of 7
isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The
14-3-3 protein identified in the cerebrospinal fluid provides a
surrogate marker for premortem diagnosis of
Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of
prion diseases remains unknown. By
protein overlay and mass spectrometric analysis of
protein extract of NTera2-derived differentiated neurons, we identified
heat shock protein Hsp60 as a 14-3-3-interacting
protein. The 14-3-3zeta and gamma
isoforms interacted with Hsp60, suggesting that the interaction is not
isoform-specific. Furthermore, the interaction was identified in SK-N-SH
neuroblastoma, U-373MG
astrocytoma, and HeLa cervical
carcinoma cells. The cellular
prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain
protein extract. By
protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning
amino acid residues 27-287 and the NTF of PrPC spanning
amino acid residues 23-137. By immunostaining, the
14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the
14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of
prion diseases.