Fleischer BR, Mackler SA

University of Pennsylvania, Veterans Affairs Medical Center, Department of Medicine

ABSTRACT

NAC-1 mRNA levels were selectively increased in the rat nucleus accumbens, a brain structure critical for drug self-administration, three weeks after chronic cocaine self-administration (Cha et al., 1997). The NAC-1 protein has an amino-terminal protein-protein interaction domain termed POZ [observed in other poxvirus and zinc finger family proteins]. POZ proteins interact with a diverse array of proteins. The POZ domain in other proteins has been shown to interact both with non-POZ proteins such as SMRT (Dhordain et al. 1997) and with POZ proteins such as Bcl-6 (Dhordain et al. 1997) and PLZF (Grignani et al. 1998). We hypothesized that NAC-1 affects gene transcription by binding to other POZ proteins in the brain. To test for PLZF and NAC-1 interactions, a co-immunoprecipitation was performed. Radiolabeled (pC17a.3b, DpC17a.3b) proteins with a (His)₆ epitope tag at their carboxy terminus, were incubated with radiolabeled PLZF. Following this, a monoclonal antibody against the (His)₆-tag was used in the immunoprecipitation. The results showed that PLZF was pulled down with either of the NAC-1 proteins. These results indicate that PLZF and NAC-1 interact independently of the NAC-1 POZ domain.

Figure 1. Gel shows three in vitro translations on left and immunoprecipitation of PLZF on right with both NAC-1 and DNAC-1

The results indicate that NAC-1 interacts in vitro with PLZF through a non-POZ domain mechanism(Fig 1). Further experiments must pursue the nature of this interaction and whether it recurs between NAC-1 and other mammalian brain proteins

The co-immunoprecipitation result calls into question the purpose of POZ domain's dimerization property and how that represses transcription. Future experiments will be aimed at determining the necessity of the POZ domain for their interactions. We will pursue more detailed structure/function studies to see which regions of NAC-1 mediate the interaction with the studied proteins. Gel supershifts may be used to identify the same proteins from brain extracts, to confirm that the NAC-1 protein is present in vivo.

Huge thanks to my P.I. Dr. Scott Mackler for all his help, patience, and wisdom.

REFERENCES

Cha X-Y, Pierce RC, Kalivas PW, Mackler SA (1997) NAC-1, a rat brain mRNA, is increased in the nucleus accumbens three weeks after chronic cocaine self-administration. J Neurosci. 17:6864-6871.

Dhordain P, Leprince D. (1997) Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci, USA. 94:10762-10767.

Gringani F, Minucci S & Pelicci PG. (1998) Fusion proteins of the retinoic acid receptor-a recruit histone deacetylase in promyelocytic leukaemia. Nature. 391:815-818.