Phosphorylation Motifs Regulating the Stability and Function of Myocyte Enhancer Factor 2A

Abstract

The phosphorylation status of the myocyte enhancer factor 2 (MEF2) transcriptional regulator is a critical determinant of its tissue-specific functions. However, due to the complexity of its phosphorylation pattern in vivo, a systematic inventory of MEF2A phosphorylation sites in mammalian cells has been difficult to obtain. We employed modern affinity purification techniques, combined with mass spectrometry, to identify several novel MEF2 phosphoacceptor sites. These include an evolutionarily conserved KSP motif, which we show is important in regulating the stability and function of MEF2A. Also, an indirect pathway in which a protein kinase casein kinase 2 phosphoacceptor site is phosphorylated by activation of p38 MAPK signaling was documented. Together, these findings identify several novel aspects of MEF2 regulation that may prove important in the control of gene expression in neuronal and muscle cells. Myocyte enhancer factor 2 (MEF2)1 is a transcriptional regulatory complex mediating diverse cellular functions in neurons (1, 2), skeletal (reviewed in Ref.3) and cardiac muscle (4 -6), and T cells (7,8). It is now well established that MEF2 plays a role in the differentiation of these cell types as well as functioning in a protective role against neuronal apoptosis.To respond to diverse developmental and physiological cues, MEF2 is structurally organized to receive and respond to multiple signals from several intracellular signaling pathways (reviewed in Refs. 3 and 9). In this regard, perhaps the best characterized is the p38 MAPK-MEF2 axis, in mammals (10, 11) and in yeast (12), although other kinase-catalyzed cascades mediated by big MAP kinase (13,14), protein kinase C (10), and protein kinase CK2 (15) are known to target MEF2. Moreover, consistent with its role as a signal sensor, putative phosphoacceptor motifs in the carboxyl terminal MEF2 transactivation domain may prove to further modulate MEF2 function in response to extracellular cues.Given that MEF2, and the biological processes it regulates, are intrinsically governed by MEF2 phosphorylation status, we undertook to systematically document MEF2 phosphorylation patterns in mammalian cells; previous phosphopeptide mapping studies used in vitro phosphorylated MEF2 protein. The purpose thus being to detect physiologically relevant, and possibly novel, in vivo MEF2 phosphorylation sites. To accomplish this we used several state-of-the-art mass spectrometric techniques to detect phosphorylation sites from MEF2 expressed in mammalian cells. To this end, we have made use of a mammalian tandem affinity purification (TAP) method (16, 17) for low-abundance nuclear transcription factors that allows purification to homogeneity and provides amounts compatible with mass spectrometric analysis of phosphorylation sites.In these studies, we have identified two important and novel aspects of MEF2 regulation. One is a highly conserved phosphoacceptor motif that regulates MEF2 stability and function. The second is an indirect pathway of MEF2 regulation by p38 MAPK me...

Satellite Cells and the Muscle Stem Cell Niche

“…The function of p38-␣/␤ is at least partially responsible for the phosphorylation of Mef2s as inhibition of p38-␣/␤ disrupts the transcriptional activities of Mef2s. In contrast, the expression of constitutively active forms of p38-␣/␤ promotes myogenesis (117,221,390,420,571,589). p38-␣/␤ activity stimulates the binding of MyoD and Mef2s to the promoters of muscle-specific genes, leading to the recruitment of chromatin remodeling complexes, and ultimately the RNA polymerase II holoenzyme (405,424,491).…”

Mechanotransduction in skeletal muscle

“…Cn dependent dephosphorylation of MEF2 appears to increase the rate of MEF2 dependent transcription (154), as does p38/PKC/CaMK dependent phosphorylation (158). It appears that an intricate, residue specific pattern of MEF2 phosphorylation (160) modulates its interaction with class II histone deacetylases (HDAC) (161) and NFAT (162), and its transcriptional activity. Calcineurin certainly plays an important role in muscle development, hypertrophy, and mechanical signaling (155,156,163,164), although the nature of that role is clouded by the complexity of its effectors (165,166).…”

Alternative Pre-mRNA Splicing Governs Expression of a Conserved Acidic Transactivation Domain in Myocyte Enhancer Factor 2 Factors of Striated Muscle and Brain

“…The Ser residue of MEF2A ␤ can be phosphorylated according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (89). This phosphoserine was detected in a MEF2A fusion protein isolated from cells in which p38 MAPK signaling was activated.…”