The yin and yang of MeCP2 phosphorylation.

S tudies of the childhood neurological disorder Rett syndrome and methyl-CpG-binding protein 2 (MeCP2) taught us that MeCP2 performs a balancing act in modulating neurological functions. Rett syndrome, characterized by cognitive deficits, motor impairments, autistic-like features, seizures, and stereotyped repetitive hand movements is caused by loss of MeCP2 function (1). On the other hand, a syndrome with overlapping features including autism, mental retardation, seizures, motor impairments, and repetitive movements results from gain of MeCP2 function due to doubling or tripling the protein level (2–4). In animal models, neuronal loss of MeCP2 leads to reduced glutamatergic synaptic strength due to reduced synapse numbers and gain of MeCP2 in neurons results in increased synaptic strength due to increased synapse numbers (5). Until recently it was believed that MeCP2 was exclusively expressed in neurons in the central nervous system (6, 7), we now know MeCP2 is also expressed in astrocytes and that MeCP2 deficient astrocytes cannot support neuronal dendritic arborization (8). At the molecular level, several studies have shown that MeCP2 functions as a transcriptional repressor by binding to methylated CpG dinucleotides and recruiting co-repressor proteins to silence gene expression (9, 10). However, in vivo studies showed that loss of MeCP2 leads to reduced expression of thousands of genes suggesting that MeCP2 may be a transcriptional modulator important for decreasing the expression of some genes and enhancing the expression of others (11). In this issue of PNAS, we learn about yet other balancing forces in modulating MeCP2 function: the differential phosphorylation of MeCP2 in response to neuronal activity (12). Such phosphorylation events may be one key mechanism by which MeCP2 modulates gene expression. Protein phosphorylation is an important posttranslational modification that can modulate the function of a protein via the addition of a phosphate group to serine, tyrosine, or threonine residues. Prior studies showed that depolarizing cultured neurons with potassium chloride (KCl) led to reduced MeCP2 association with the promoter of Brain derived neurotrophic factor (Bdnf ) and a corresponding increase in Bdnf transcription (13, 14). Zhou and colleagues identified that the activity-dependent phosphorylation of serine 421 (S421) in MeCP2 leads to transcriptional induction of Bdnf (15). Together, these studies provided the initial evidence suggesting that phosphorylation of MeCP2 integrates neuronal activity with transcription of a target gene. The new study by Tao et al. achieves a key milestone in elucidating the dynamic balance between site-specific dephosphorylation and phosphorylation that enables MeCP2 to control transcription of specific target genes (12). The authors surveyed phosphorylated serine, threonine, and tyrosine residues of rat and mouse MeCP2. They determined that these residues are all conserved, but not necessarily similarly phosphorylated across species. Further analysis in mouse brain samples revealed that serine 80 (S80) and serine 399 are the two major phosphorylation sites under resting conditions. Two residues showed specific activity-dependent phosphorylation, serine 424 (S424) and the previously identified S421. Tao and colleagues discovered that neuronal activity-induced calcium influx through L-type voltage gated calcium channel triggers calcium/calmodulindependent protein kinase IV (CamK IV) to phosphorylate S421. When either neuronal activity or calcium influx is blocked pharmacologically, S421 is dephosphorylated. In contrast to S421, S80 is the most constitutively phosphorylated residue in resting neurons and is dephosphorylated with neuronal activity. Based on the S421 results, could S80 dephosphorylation be mediated by a phosphatase, such as calcineurin that is found in the highest concentrations in the brain? The phosphatase activity of calcineurin increases in response to calcium influx (16), thus it would be interesting to determine whether calcineurin inhibitors such as cyclosporin and tacrolimus inhibit the activity-dependent dephosphorylation of S80. To explore the in vivo biological consequences of the key phosphorylation events, the authors generated two phosphorylation deficient knockin mouse models, one carrying a single mutant, S80A MeCP2, and the other expressing a MeCP2 with two mutations, S421A/ S424A, to demonstrate that the phosphorylation of S80, S421, and S424 have biological consequences in vivo. Interestingly, mice expressing the single mutant S80A exhibit weight gain and decreased locomotor activity, which is suggestive of possible decreased MeCP2 function. Hypoactivity and weight gain are observed in various mouse models that have complete or partial loss of MeCP2 function (17–21). In contrast, mice carrying the S421A/S424A double mutation have normal weight and in-