Isolation of hNap1BP which interacts with human Nap1 (NCKAP1) whose expression is down-regulated in Alzheimer's disease
Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder and the etiology of AD is very complex. To date, three genes, APP (Goate et al., 1991) and the Presenilins (PS1 and PS2) (Sherrington et al., 1995, Levy-Lahad et al., 1995), involved in familial AD (FAD) have been identified. However, these genes are not sufficient to account for the onset of AD in general. Therefore, it is important to identify and analyze genes associated with sporadic AD in order to elucidate the general onset of AD. We previously reported the identification of the human Nap1 gene which was markedly decreased in sporadic AD (Suzuki et al., 2000) and human Nap1 was shown to be an orthologue of rat Nap1 (Kitamura et al., 1996). Rat Nap1 was originally identified as a binding protein to the first Src homology 3 (SH3) domain of Nck (Kitamura et al., 1996) which is an adaptor molecule containing one SH2 domain and three SH3 domains and can influence cytoskeletal events (Bagrodia and Cerione, 1999). Rat Nap1 is a member of the HEM family, conserved to a high degree from invertebrates through to mammals (Baumgartner et al., 1995). Rat Nap1 was not only associated with Nck (Kitamura et al., 1996) but also with the small GTPase Rac1 (Kitamura et al., 1997) which is an important regulator of the actin cytoskeleton (Hall, 1998) and Profilin II (Witke et al., 1998) which participates in actin dynamics. KETTE, a member of the HEM family in Drosophila melanogaster, has an essential role in cytoskeletal organization during axonal path-finding (Hummel et al., 2000). Mutations in the kette gene affect the organization of the cytoskeleton. Besides cytoskeletal signaling, we have previously shown another novel function of the HEM family which is that the suppression of human Nap1 transcripts induced apoptosis in neuroblastoma cell lines (Suzuki et al., 2000). Since one of the major pathological features of AD is a loss of neurons by neuronal cell death, human Nap1 may play a role in neuronal cell death in AD. Therefore, it is significant to isolate molecules which interact with human Nap1 in order to begin to understand the biologically fundamental process leading to AD. Here we report the identification of hNap1BP which interacts with human Nap1. hNap1BP was highly homologous to the tyrosine kinase-binding protein family which binds to c-Abl, a protein involved in apoptosis (Yuan et al., 1997, Yuan et al., 1999) and cell cycle arrest induced by DNA damage (Yuan et al., 1996), and the Nck adaptor molecule which is involved in multiple signaling networks (Li and She, 2000).
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hNap1/T-vec and hNap1 Cter/T-vec
To obtain the fragments encoding full ORF of human Nap1 and C terminus (amino acids 871–1128), PCR was performed using EcoRI site containing primers 5′-CCGGAATTCTCCACCATGTCGCGCTCAGTG-3′ and 5′-GGAATTCTTATGCAGAAGATGTAACACTTTG-3′, 5′-GGAATTCGTTGTGGTGGAGAATGTTGATGTG-3′ and 5′-GGAATTCGCTTATGCAGAAGATGTAACACTTTG-3′. As a template, positive phage containing full ORF of human Nap1 was used. PCR products were cloned into pT7 Blue Vector (Novagen).
Nck 1st SH3/T-vec, Nck 2nd SH3/T-vec and Nck 3rd SH3/T-vec
To obtain the fragments encoding the first (amino acids
Identification of a molecule which interacts with human Nap1 by the yeast two-hybrid system
Human Nap1 was an orthologue of rat Nap1 (99.2% with the amino acid sequence and 90% with the nucleotide sequence). Recently rat Nap1 was reported to interact with Nck (Kitamura et al., 1996), small GTPase Rac1 (Kitamura et al., 1997) and Profilin II (Witke et al., 1998). Therefore, we examined whether human Nap1 interacted with the human homologues of the above-mentioned molecules. Human Nap1 associated with the Nck 1st SH3 domain in our experimental system (data not shown). We considered that
Discussion
Identification of protein–protein interactions is a valuable method for defining new components of the pathway associated with human Nap1. The present study revealed that the identification of the molecule, hNap1BP, interacts with human Nap1. The expression of human Nap1 is known to be decreased in sporadic AD affected brains and the suppression of human Nap1 function is related to apoptosis (Suzuki et al., 2000). The hNap1BP gene consisted of a 2281 nucleotide sequence and was ubiquitously
Acknowledgements
We are grateful to Dr Yoshiro Maru for the generous gift of the c-Abl construct.
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