PROTEOLYTIC FRAGMENTS OF THE ALZHEIMERS-DISEASE ASSOCIATED PRESENILIN-1 AND PRESENILIN-2 ARE PHOSPHORYLATED IN-VIVO BY DISTINCT CELLULAR MECHANISMS

The majority of familial Alzheimer's disease mutations are linked to t he recently cloned presenilin (PS) genes, which encode two highly homo logous proteins (PS-1 and PS-2). Full-length PS proteins undergo endop roteolytic cleavage within their hydrophilic loop domain resulting in the formation of C-terminal (CTF) and N-terminal fragments (NTF). PS-2 was found to be phosphorylated as a full-length protein within its N- terminal domain. In contrast, PS-1 is phosphorylated selectively after proteolytic processing within its similar to 20 kDa CTF involving pro tein kinase C (PKC) and/or protein kinase A (PKA). We now have found t hat the CTF of the highly homologous PS-2 is also phosphorylated. Surp risingly, the PS-2 CTF is not phosphorylated by PKC or PKA. Instead, t he PS-2 CTF is constitutively phosphorylated in vivo by serine/threoni ne protein kinases, which are independent of phorbol ester and intrace llular cAMP. In vitro the large hydrophilic loop of PS-2 between trans membrane domains 6 and 7 can be phosphorylated by casein kinase-1 (CK- 1) and CK-2, but not by PKA or PKC. Quantitative analysis of in vitro phosphorylation demonstrates the presence of two phosphorylation sites for CK-1 and a single site for CK-2. A deletion analysis revealed tha t the CTF of PS-2 is phosphorylated in vivo within an acidic sequence containing three potential phosphorylation sites for CKs (serines 327, 330, and 335). These data suggest that CK type protein kinases phosph orylate the CTF of PS-2 within its hydrophilic loop domain in vivo. In terestingly, the potential phosphorylation sites are located directly adjacent to the recently identified caspase cleavage sites.