Phosphorylation of the cytoplasmic domain of Alzheimer's beta-amyloid precursor protein at Ser655 by a novel protein kinase.

The cytoplasmic domain of Alzheimer's beta-amyloid precursor protein (APP) can be phosphorylated at Thr654, Ser655, and Thr668 (APP695 isoform numbering). Previous studies demonstrated that Ser655 of APP was phosphorylated by protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) in vitro and by unidentified protein kinase(s) in vivo. We report here the characterization of a novel protein kinase (designated APP kinase I) which phosphorylates Ser655 of APP. APP kinase I was partially purified over 7,000-fold from rat brain and identified as a approximately 43 kDa protein that is distinct from a number of known protein kinases, including PKC and extracellular signal-regulated kinases (ERKs). The identification of a novel protein kinase that phosphorylates Ser655 will hopefully contribute to our understanding of the metabolism and/or function of APP in the pathogenesis of Alzheimer's disease (AD).

Role of phosphorylation of Alzheimer's amyloid precursor protein during neuronal differentiation.

Alzheimer's amyloid precursor protein (APP), the precursor of beta-amyloid (Abeta), is an integral membrane protein with a receptor-like structure. We recently demonstrated that the mature APP (mAPP; N- and O-glycosylated form) is phosphorylated at Thr668 (numbering for APP695 isoform), specifically in neurons. Phosphorylation of mAPP appears to occur during, and after, neuronal differentiation. Here we report that the phosphorylation of mAPP begins 48-72 hr after treatment of PC12 cells with NGF and that this correlates with the timing of neurite outgrowth. The phosphorylated form of APP is distributed in neurites and mostly in the growth cones of differentiating PC12 cells. PC12 cells stably expressing APP with Thr668Glu substitution showed remarkably reduced neurite extension after treatment with NGF. These observations suggest that the phosphorylated form of APP may play an important role in neurite outgrowth of differentiating neurons.

8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid inhibits MAPK phosphorylation in endothelial cells induced by VEGF and bFGF.

Angiogenesis is regulated by various factors. In particular, VEGF and basic FGF are of much importance. We found that 8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid inhibited the binding of VEGF to KDR/Flk-1 (VEGF receptor-2) or Flt-1 (VEGF receptor-1) and that it inhibited the MAPK phosphorylation in HUVEC induced by VEGF or basic FGF but not by EGF. 8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid might be used as an inhibitor of VEGF and basic FGF signal transduction.

Sck interacts with KDR and Flt-1 via its SH2 domain.

Vascular endothelial growth factor (VEGF) is one of the major angiogenesis regulators. It binds to its tyrosine kinase receptors, KDR and Flt-1. However, little is known about their downstream signal transduction properties. We screened human brain cDNA library using the yeast two-hybrid system with the KDR cytoplasmic region as bait to find KDR binding proteins. After 6.2 x 10(6) clones were screened, we identified Sck, one of the Shc homologues, as a KDR binding protein. Sck also binds to Flt-1 and their binding is dependent on the kinase activities of KDR and Flt-1. Extensive site-directed mutagenesis of KDR revealed that Y1175 of KDR is a major binding site for Sck. As Sck contains the SH2 domain and PTB domain, we tested whether they bind to KDR and Flt-1. The SH2 domain of Sck binds to both of them. Deletion of the SH2 domain from Sck resulted in the complete loss of binding. On the other hand, the PTB domain of Sck does not bind to KDR and Flt-1. These results indicate that Sck binds to KDR and Flt-1 via its SH2 domain and might play an important role in VEGF signal transduction.

Tyrosine 1213 of Flt-1 is a major binding site of Nck and SHP-2.

Vascular endothelial growth factor (VEGF) binds to its receptor tyrosine kinase Flt-1 and KDR/Flk-1 and stimulates their autophosphorylation. However, little is known about their downstream signal transduction properties. We examined the interactions of certain proteins with a SH2-domain with Flt-1 and KDR using the yeast two-hybrid system and found that Nck, SHP-2, PLC gamma, and PI3K p85 bind to Flt-1. Extensive site-directed mutagenesis of Flt-1 revealed their major binding sites. Nck, SHP-2, and PI3K bind to Y1213 of Flt-1. Nck also binds to Y1333 of Flt-1. These results suggest that Nck, SHP-2, PLC gamma, and PI3K play important roles in Flt-1 signal transduction and that Y1213 of Flt-1 is a major binding site of PI3K, Nck, and SHP-2.

Phosphorylation of Alzheimer beta-amyloid precursor-like proteins.

Amyloid precursor-like proteins (APLPs), APLP1 and APLP2, are members of a gene family which include the Alzheimer beta-amyloid precursor protein (APP). APLP1, APLP2, and APP contain highly homologous amino acid sequences, especially in their cytoplasmic domains, although APLPs lack the beta-amyloid domain derived by proteolytic processing from APP. APP is phosphorylated at three sites in the cytoplasmic domain in cultured cells and adult rat brain [Suzuki et al. (1994) EMBO J. 13, 1114-1122; Oishi, et al. (1997) Mol. Med. 3, 109-121] and at sites in the extracellular domain in cultured cells [Knops et al. (1993) Biochem. Biophys. Res. Commun. 197, 380-385; Hung & Selkoe (1994) EMBO J. 13, 534-542; Walter et al. (1997) J. Biol. Chem. 272, 1896-1903]. We report here that a cytoplasmic domain peptide from APLP1 is phosphorylated in vitro by protein kinase C and that a cytoplasmic domain peptide from APLP2 is phosphorylated in vitro by protein kinase C and cdc2 kinase. APLP2 is phosphorylated by cdc2 kinase at a site homologous to the cdc2 kinase site phosphorylated in APP. Furthermore, phosphorylation of this site occurs in a cell cycle-dependent manner in cultured cells. These findings indicate that in intact cells the phosphorylation of APLP2 appears to be regulated in a similar fashion to that of APP.

The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.

BACKGROUND: The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). MATERIALS AND METHODS: We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. RESULTS: We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. CONCLUSIONS: The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.

Purification and characterization of new anti-adrenocorticotropin rabbit neutrophil peptides (defensins).

Neutrophil peptides (NPs, defensins), which consist of approximately 30 amino acids with a highly conserved backbone of six Cys residues, possess several biological activities, such as antimicrobial, antiviral, cytotoxic, and anti-adrenocorticotropin (corticostatic) activity in vitro. In the rabbit, six NPs, i.e., NP-1, -2, -3A, -3B, -4, and -5, have been isolated, among them, NP-3A has the most potent corticostatic activity, which involves the inhibition of adrenocorticotropin-stimulated corticosterone synthesis in adrenal cells. Using a sensitive radioimmunoassay for NP-3A and reverse-phase HPLC, we found a novel component in rabbit tissue extracts with NP-3A-like immuno-reactivity. We further purified and characterized the component and found two novel peptides. One of these peptides, designated NP-6, has an amino acid sequence identical to that of NP-3A except for three amino acids, i.e. Leu11, Phe13, and Gln15, in the central portion of the sequence. The other has a sequence corresponding to that of NP-6 except that the N-terminal Gly was deleted and is thus named des-G1-NP-6. Using chemically synthesized NP-6 and des-G1-NP-6, we measured their putative corticostatic activities by a dispersed rat adrenal cell bioassay. NP-6 showed corticostatic activity comparable to that of NP-3A while des-G1-NP-6 showed weak activity. These findings are also compatible with the notion that the N-terminal Gly, but not the central portion, is important for the anti-adrenocorticotropin activity of the NPs.