Casein Kinase II Phosphorylates the Fragile X Mental Retardation Protein and Modulates Its Biological Properties

Abstract

Fragile X syndrome is caused by loss of FMR1 protein expression. FMR1 binds RNA and associates with polysomes in the cytoplasm; thus, it has been proposed to function as a regulator of gene expression at the posttranscriptional level. Posttranslational modification of FMR1 had previously been suggested to regulate its activity, but no experimental support for this model has been reported to date. Here we report that FMR1 in Drosophila melanogaster (dFMR1) is phosphorylated in vivo and that the homomer formation and the RNAbinding activities of dFMR1 are modulated by phosphorylation in vitro. Identification of a protein phosphorylating dFMR1 showed it to be Drosophila casein kinase II (dCKII). dCKII directly interacts with and phosphorylates dFMR1 in vitro. The phosphorylation site in dFMR1 was identified as Ser406, which is highly conserved among FMR1 family members from several species. Using mass spectrometry, we established that Ser406 of dFMR1 is indeed phosphorylated in vivo. Furthermore, human FMR1 (hFMR1) is also phosphorylated in vivo, and alteration of the conserved Ser500 in hFMR1 abolishes phosphorylation by CKII in vitro. These studies support the model that the biological functions of FMR1, such as regulation of gene expression, are likely regulated by its phosphorylation.Fragile X syndrome is the most frequent cause of inheritable mental retardation and is also one of the most common singlegene disorders (14). Cognitive deficits reported for fragile X children range from mild to severe, and behavioral disturbances include social and attention deficits, autistic-like behaviors, unusual responses to sensory stimuli, hyperactivity, and sleep problems (12,14). The gene directly responsible for fragile X syndrome, FMR1, was identified in 1991 (47). In most cases, the syndrome is caused by a trinucleotide repeat expansion in the 5Ј untranslated region of the FMR1 gene (49). The expansion of the CGG repeat results in an absence of the encoded protein. It is therefore clear that the pathophysiological mechanisms leading to symptoms in fragile X syndrome can be elucidated by studying the functions of the FMR1 gene.The fact that the FMR1 protein is a cytoplasmic protein with RNA-binding motifs (two KH domains and an RGG box) (2, 38, 39) and the fact that it associates with ribosomes (6, 9, 21, 41) suggest that this protein functions in the posttranscriptional regulation of some specific mRNA expression (16). Indeed, FMR1 has recently been proposed to be a translational repressor in a cell-free system and in frog oocytes (26,27). Interestingly, although the inhibitory effect was demonstrated not to be influenced by the features of the mRNAs used in those studies, FMR1 seemed to specifically inhibit its own translation (36). It has been known that FMR1 is found at postsynaptic sites enriched in ribosomes, where some specific mRNAs, including FMR1, are translated in response to synaptic activation (13). It has also been observed that in fragile X patients the numbers of dendritic spines are different and the mo...

S6K1 Phosphorylates and Regulates Fragile X Mental Retardation Protein (FMRP) with the Neuronal Protein Synthesis-dependent Mammalian Target of Rapamycin (mTOR) Signaling Cascade

“…FMRP is phosphorylated on a highly conserved serine residue, and it has been previously shown in Drosophila that Drosophila FMRP (dFMRP) is phosphorylated by casein kinase II (38). However, we failed to detect any effect of casein kinase II on FMRP phosphorylation in primary mammalian neurons in the presence of mGluR activity (Fig.…”

New insights into fragile X syndrome: from molecules to neurobehaviors

“…However, the type of modification had not been identified until recently [32]. Now, it has been shown that both human FMRP and fly dfmr1 protein are phosphorylated in vivo [33]. The phosphorylation site is conserved through evolution at Ser406 in dfmr1 protein and Ser500 in human FMRP.…”

“…In addition, Drosophila casein kinase II (dCKII) directly interacts with, and phosphorylates, dfmr1 protein in vitro. The alteration of the conserved serine residue in both FMRP and dfmr1 protein abolishes phosphorylation by CKII in vitro [33]. However, it will be important to determine which kinase phosphorylates FMRP and dfmr1 protein in vivo, and how the phosphorylation regulates FMRP activity in vivo.…”

Fragile X Mental Retardation Protein Induces Synapse Loss through Acute Postsynaptic Translational Regulation

“…Like CPEB and ZBP, phosphorylation of FMRP on a conserved serine (406 in Drosophila, 499 in mouse, and 500 in human) has been suggested to be a mechanism to regulate translational suppression and activation by FMRP. In vitro phosphorylation of Drosophila FMR affected binding to poly U, but not poly G RNA (Siomi et al, 2002). However, mimicking phosphorylation of murine FMRP at Ser499 with Asp does not affect binding to known target mRNAs, but rather increases association of FMRP with stalled polysomes in vivo.…”