The known mammalian PP1 regulators fall into two categories, targeting subunits and inhibitor proteins. Many members of these two groups of proteins possess a conserved KIXF motif, a pivotal binding site for the PP1 catalytic subunit (16
). Consistent with the competition among these protein regulators for binding the KIXF docking pocket in the PP1 catalytic subunit, PP1 bound to the prototypic PP1-targeting subunit GM
and its structural homologue PTG (protein targeting to glycogen) and shows decreased sensitivity to the inhibitors I-1 (38
) and DARPP-32 (7
), and dissociation of the PP1 catalytic subunit from these targeting subunits enhances PP1 regulation by these inhibitors. Thus, endogenous PP1 inhibitors such as CPI-17 (48
), which lacks a KIXF motif, are considered more viable mechanisms for regulating PP1 holoenzymes, such as the smooth muscle myosin phosphatase (58
). However, recent studies (23
) have identified two PP1 holoenzyme inhibitors (PHIs). PHI-I, although structurally related to CPI-17, contains a putative RVXF motif and inhibits the glycogen-bound and myosin-bound PP1 complexes. This suggests novel mechanisms of PP1 regulation that can circumvent the potential competition between targeting subunits and inhibitors for the KIXF binding pocket on the PP1 catalytic subunit.
I-1 activity as a PP1 inhibitor is increased more than 25,000-fold following its phosphorylation by PKA (19
), making I-1 a remarkable signal transduction switch or gate that amplifies cAMP signals in many mammalian cells. Structure-function studies of I-1 (19
) and its structural and functional homologue DARPP-32 have established that the key elements for PP1 regulation are localized in the highly homologous N-terminal 50 amino acids, which contain not only the PKA-phosphorylated threonine but also the KIXF motif required for PP1 inhibition by these proteins. The role of their unique C-terminal sequences remains largely unknown. The finding that I-1 forms a stable association, at least in vitro, with the growth arrest and DNA damage-inducible protein GADD34 provided the first evidence for a novel function of the I-1 C terminus, namely, to recruit cellular proteins that modulate the PP1 catalytic subunit. Thus, GADD34 established a new regulatory paradigm that brings two distinct KIXF-containing PP1 regulators, a targeting subunit and an inhibitor, to the same PP1 complex.
Structure-function analysis of I-1 showed that the C-terminal 30 amino acids of I-1 were not essential for PP1 binding or inhibition but were required for GADD34 binding. Conversely, analysis of GADD34 suggested that PP1 and I-1 may bind different regions of this protein, with PP1 binding to a C-terminal domain that is conserved in the viral proteins HSV-1 ICP34.5 and avian sarcoma virus (ASV) NL-S, which harbor a KIXF PP1-binding motif (34
), and I-1 binding to the central domain of GADD34, which contains multiple 34-amino-acid repeats. Finally, enzymatic studies showed that, unlike PP1 bound to GM
, the prototypic PP1 targeting subunit, which displays a reduced sensitivity to I-1 (38
), the presence of excess GADD34 did not preclude PP1 inhibition by PKA-phosphorylated I-1. Together, these studies pointed to independent interactions of I-1, PP1, and GADD34 through adjacent but separable sites on the individual proteins, with the potential to form a heterotrimeric I-1/GADD34/PP1 complex. This raised the intriguing possibility that by scaffolding of PP1 and I-1 together, the requirement for the KIQF sequence in I-1 for PP1 inhibition may be eliminated, as the two key elements necessary for PP1 inhibition (19
), the phosphorylated threonine and the KIXF motif, are provided by two different closely associated PP1 regulators, I-1 and GADD34, respectively. Alternately, the GADD34-bound I-1 may be subject to the physiological phosphorylation at serine-67 catalyzed by the neuronal protein kinase Cdk5 (4
), which regulates neuronal differentiation and development.
Early studies used reticulocyte lysates as a model system for protein synthesis and showed that phosphorylated I-1 (and I-2, another PP1-specific inhibitor) inhibited protein synthesis, specifically the formation of the translation initiation complex, by increasing eIF-2α phosphorylation (22
). As PP1 inhibitors did not alter eIF-2α kinase activity (55
), this identified eIF-2α as the PP1 substrate. Genetic antagonism between GLC7, the gene encoding the yeast PP1 catalytic subunit, and GCN2, the eIF-2α kinase, also pointed to a conserved function for PP1 as an eIF-2α phosphatase (66
). In reticulocyte lysates (21
), increases in cAMP activated PKA and inhibited the assembly of the translation initiation complex. PKA did not directly phosphorylate eIF-2α or hemin-regulated inhibitor (also known as HCR), the eIF-2α kinase present in reticulocytes and other cells. Thus, it was speculated that cAMP may mediate its effects through a reduction in eIF-2α phosphatase activity. However, the precise mechanism by which cAMP inhibited eIF-2α phosphatase has not been resolved. Thus, it is tempting to speculate that PKA phosphorylates the GADD34-bound I-1 to suppress the eIF-2α phosphatase activity of the PP1 catalytic subunit bound at the neighboring site on GADD34 and represents a mechanism for cAMP inhibition of protein synthesis in mammalian tissues.
In pursuing the physiological role of GADD34, overexpression of full-length GADD34 (1
) has been shown to promote apoptosis in tissue culture cells. Similarly, nutrient deprivation, UV and gamma irradiation, anticancer drugs (43
), and other inducers of GADD34 transcription have varied and damaging effects on cultured cells. Thus, we used the experimental model of hibernating ground squirrels, which demonstrate a reversible shutoff of global protein synthesis associated with hyperphosphorylation of eIF-2α (30
). Our studies showed that hibernation is another cell stress that induces GADD34 levels in brain tissue. While the reduced metabolic activity and blood flow in the hibernating squirrel brain resulted in dephosphorylation of threonine-35 and inactivated I-1, eIF-2α phosphorylation was greatly increased. Analysis of I-1/GADD34 and PP1/GADD34 interactions showed that both PP1 and I-1 bound GADD34 in brain tissue from active animals, which demonstrated low steady-state phosphorylation of eIF-2α. In contrast, despite the elevated expression of GADD34 in hibernating brain tissue, its associations with both PP1 and I-1 were disrupted. Coincident with the loss of these protein-protein interactions, eIF-2α phosphorylation was elevated and protein synthesis was inhibited in the hibernating squirrel brain. This argued that the GADD34-bound phosphatase complex played a key role in the control of protein translation and suggested that changes in I-1 phosphorylation/activity, altered GADD34 expression, and coordinated recruitment of I-1 and PP1 to the GADD34 protein may represent distinct mechanisms for controlling protein synthesis in the mammalian brain following various forms of cell stress.
Studies of the mechanisms underlying host cell infection by HSV-1 pointed to a key role for ICP34.5, the product of the γ134.5
gene, in viral infectivity of neuronal cells. Analysis of extracts from cells infected with HSV-1 expressing a functional γ134.5
gene showed that its gene product, ICP34.5, bound PP1 and increased eIF-2α phosphatase activity by nearly 3,000-fold (35
) compared to uninfected cells or cells infected with HSV-1 containing a mutant γ134.5
gene. Thus, the expression of ICP34.5 represented a mechanism by which HSV-1 precluded host cell-mediated shutoff of protein synthesis. Structural homology between HSV-1 ICP34.5 and the C-terminal region of GADD34 and the ability of this region of the mouse GADD34 homologue, Myd116 (49
), to complement the loss of the viral γ34.5 gene argued that PP1 bound to the GADD34 C terminus may also function as an eIF-2α phosphatase. In this regard, our biochemical studies established that GADD34(230–674) had the hallmarks of a regulatory or targeting subunit that reduced or abolished PP1 activity against phosphorylase a
but was an active eIF-2α phosphatase. This provided experimental support for the notion that the cellular GADD34/PP1 complex, like the ICP34.5/PP1 complex, may also regulate protein synthesis.
However, ICP34.5 differs from GADD34 in that it does not induce apoptosis but rather favors cell survival in mammalian cells. Whether this represents differences in the recruitment of other proteins by the viral and cellular PP1-binding proteins or differences in biochemical properties of the eIF-2α phosphatases assembled by these proteins is currently unknown. For example, the C-terminal domain of ICP34.5 that recruits PP1 also binds the cell cycle protein PCNA (9
). In contrast, PCNA binding to GADD34 has not been demonstrated. Interestingly, ICP34.5 expression inhibits apoptosis induced by GADD34 (59
), possibly suggesting that they compete for common cellular targets. In this regard, differences in PP1 binding and regulation by ICP34.5 and GADD34, which may contain both positive and negative regulatory elements, may also account for their different cellular effects. While increases in eIF-2α phosphorylation have been linked to programmed cell death (62
), it is also possible that the apoptotic effects of GADD34 reflect other protein interactions or differences in turnover of eIF-2α phosphorylation at serine-51 by the GADD34-bound PP1 compared to the ICP34.5-bound PP1. A clear distinction between these two proteins is the unique ability of GADD34 to recruit I-1, a known PP1 regulator. Understanding the functional interactions between PP1 and I-1 within the GADD34 signaling complex and their regulation by physiological signals should yield new insights into the physiological role of the complex in the control of protein synthesis and the choice between apoptosis and cell survival.
In addition to PP1 and I-1, several other GADD34-binding proteins have been identified, including the transcription factor BFCOL1, which binds to the promoter of the gene encoding the p21cdk
), KIF1A kinesin (33
) and the product of the HRX gene that is translocated in many human leukemias (1
). All of these proteins associate with the central region of GADD34 that also binds I-1. This raises the possibility that I-1 also competes with cellular proteins for GADD34 binding and suggests a complex regulation of the GADD34-bound phosphatase. As HRX association attenuates GADD34's ability to induce apoptosis, and it may displace I-1 or other GADD34-bound proteins that participate in apoptotic signaling. Finally, rat cell lines express a proliferation marker, PEG3, with sequence homology to the N terminus and central domain of GADD34 but does not contain a PP1-binding domain (64
). Whether PEG3 is expressed in other species and contributes to regulation of the I-1/GADD34/PP1 complex requires further investigation.
Identification of numerous GADD34-binding proteins suggests a wider role for GADD34 than simply the regulation of protein translation. In this regard, analysis of genetically modified mice, such as the I-1-null mice (3
), which display a complex neuronal phenotype, may provide insights into the specific contribution of I-1, and perhaps PP1, in eIF-2α dephosphorylation and protein translation. In summary, we have identified the first PP1 complex that may contain more than one PP1 regulator, and the challenge for future studies is to elucidate the cell signaling events that modify the assembly and activity of this signaling complex to control protein synthesis, cell survival, and apoptosis.