The Human SNF5/INI1 Protein Facilitates the Function of the Growth Arrest and DNA Damage-inducible Protein (GADD34) and Modulates GADD34-bound Protein Phosphatase-1 Activity

Abstract

The growth arrest and DNA damage-inducible protein (GADD34) mediates growth arrest and apoptosis in response to DNA damage, negative growth signals, and protein malfolding. GADD34 binds to protein phosphatase-1 (PP1) and can attenuate translational elongation of key transcriptional factors through dephosphorylation of eukaryotic initiation factor-2␣. We reported previously that the human trithorax leukemia fusion protein (HRX) can bind to GADD34 and abrogate GADD34-mediated apoptosis in response to UV irradiation. We found that hSNF5/INI1, a component of the hSWI/SNF chromatin remodeling complex, also binds to GADD34 and can coexist with GADD34 and HRX fusion proteins as a trimolecular complexes in vivo. In the present report, we demonstrate that hSNF5/INI1 binds to GADD34 in part through the PP1 docking site within a domain homologous to herpes simplex virus-1 ICP34.5. We found that hSNF5/INI1 can bind PP1 independently and weakly stimulate its phosphatase activity in solution and in complex with GADD34. hSNF5/INI1 and PP1 do not compete for binding to GADD34 but rather form a stable heterotrimeric complex with GADD34. We also show that Epstein-Barr nuclear protein 2, which binds hSNF5/INI1, can disrupt hSNF5/INI1 binding to GADD34 and partially reverse the GADD34-mediated growth suppression function in Ha-ras expressing HIH-3T3 (3T3-ras) cells. These results implicate hSNF5/INI1 in the function of GADD34 and suggest that hSNF5/INI1 may regulate PP1 activity in vivo. GADD341 is a growth arrest and DNA damage-inducible gene (1), whose level of transcript is increased in response to a variety of agents that elicit genomic damage and apoptosis (2, 3) as well as by amino acid deprivation and by agents that lead to protein malfolding in the endoplasmic reticulum (4). The transcriptional regulation of GADD34 has been shown to be independent of functional p53 (2). At its carboxyl terminus, GADD34 harbors a highly conserved domain homologous to ICP34.5 of HSV1, a virulence factor that blocks the premature shut off of protein synthesis in HSV1-infected neuroblastoma cells and may interfere with apoptosis (5, 6). Although the homologous domain of murine GADD34 (MyD116) can supply the anti-apoptotic functions of viral ICP34.5 (7), mammalian GADD34 likely functions to facilitate both growth arrest and apoptosis in response to DNA damage and other cellular stresses (3,8,9).Recently both viral ICP34.5 and mammalian GADD34 proteins have been shown to regulate the activity of protein phosphatase 1 (PP1) in vitro (10, 11). ICP34.5 coexists in a high molecular weight complex with PP1 in cellular extracts derived from HSV1-infected HeLa cells (10). Mutations in either ICP34.5 or GADD34 which disrupt their binding to PP1 also impair PP1 activity and the protein malfolding response (4, 12). Recent evidence suggests that the translational elongation factor eIF2␣ is a likely biological substrate for PP1 (10 -12). eIF2␣ can be phosphorylated by several serine/threonine kinases, including the double-stranded RNA-activated kinase that i...

Gadd34 functional domains involved in growth suppression and apoptosis

“…This does not rule out the effects of viral homology deletions on other parameters such as transformation, and complex effects such as tumor formation and angiogenesis in whole animals. Recently, it was shown that binding of Gadd34 to SNF5 was required for Gadd34-mediated growth suppression since Gadd34 mutations that prevented SNF5 binding also prevented growth suppression (Wu et al, 2002). SNF5 binding was shown to be nearer the carboxyl end of Gadd34 (see Figure 4), suggesting that SNF5 may permit Gadd34-mediated growth suppression through exlusion of other Gadd34-binding proteins whose binding to Gadd34 prevents these effects.…”

“…For example, PP1, Snf5, Lyn kinase and HRXbinding domains have all been mapped to this region of Gadd34 (Figure 4), and interaction of Gadd34 with these proteins has been reported to be necessary for Gadd34-mediated growth suppression and apoptosis (Adler et al, 1999;Grishin et al, 2001;Novoa et al, 2001;Wu et al, 2002). In addition, HRX, G34BP, GaHSP40 and Translin all bind the carboxyl half of the protein, which includes the region missing in PEG-3 (Adler et al, 1999;Isobe, 1999, 2000;Hasegawa et al, 2000a, b).…”

“…Gadd34 binds protein phosphatase type 1 (PP1), and can modulate its activity and substrate specificity (Novoa et al, 2001). Gadd34 binds a number of other proteins, some of which are known modulators of protein phosphatase activity also, such as SNF5/INI1 (Wu et al, 2002) and inhibitor 1 (I1) (Connor et al, 2001). Notably, Gadd34 binds HRX, a protein overexpressed as a result of translocation/gene fusion in lymphoid and myeloid leukemia (Adler et al, 1999).…”

Members of the hSWI/SNF chromatin remodeling complex associate with and are phosphorylated by protein kinase B/Akt

“…Phosphatidyl inositol 4,5-bisphosphate is also involved in the activation of the serine/ threonine kinase Akt, by recruiting it to the plasma membrane, where it is activated via phosphorylation. In addition, INI1 was recently shown to interact with the catalytic subunit of protein phosphatase 1 (Wu et al, 2002), which has a multitude of substrates, including Akt (Xu et al, 2003). These observations led us to question whether a potential relationship also existed between Akt and the hSWI/SNF complex.…”

The Protein Phosphatase-1 (PP1) Regulator, Nuclear Inhibitor of PP1 (NIPP1), Interacts with the Polycomb Group Protein, Embryonic Ectoderm Development (EED), and Functions as a Transcriptional Repressor

“…6). Similarly, PP1 was identified as a component of another chromatin-remodeling complex, which also contains the PP1 interactor GADD34 and the SNF5 protein (43). GADD34 is a stress-induced protein that facilitates cell-cycle arrest, whereas SNF5 is a component of a SWI/SNF chromatin remodeling complex that acts by repositioning nucleosomes.…”