Ca2+ transient‐dependent changes in embryonic myocyte proteins during myofibrillogenesis a proteomic analysis

Abstract

Ca2+ transients regulate myofibrillogenesis, probably by mediating protein expression and phosphorylation changes. In the present study, Ca+2 transients in developing Xenopus embryos were blocked by ryanodine. A proteomic analysis was conducted by running a series of 2D gels using sequential extraction procedures of increasing stringency which included 1) osmotic lysis, 2) 1% TX-100, and 3) O Farell s buffer. 2D gels produced from the sequential extracts resulted in spot patterns of manageable complexity. Spots of interest were excised and identified by mass spectrometry (MS). The majority of cytosolic and un-incorporated sarcomeric proteins were resolved in extracts 1 and 2. Myosin RLC (MLC2f) and ELC (MLC1f/3f), as well as other less soluble sarcomeric proteins, were resolved using extract 3. Several spot pairs in each extract show significant ryanodine-sensitive pI shifts, which are consistent with differences in phosphorylation state. One pair of particular interest was identified as MLC3f, a myosin essential light chain known to be an early marker of somitic muscle differentiation in Xenopus laevis embryo. Our results suggest that MLC3f is phosphorylated in controls but not in ryanodine treated embryos. To our knowledge this is the first study to show specific phosphorylation of the essential light chains of myosin. In order to extend and verify these results, a complimentary approach based on fluorescence 2D differential gel electrophoresis (DIGE) was also conducted. To verify that spot shifts are due to Ca+2 transient-mediated phosphorylation and identify the specific residue(s), phosphopetides from spots of interest will be isolated by immobilized metal affinity chromatography (IMAC) and analyzed by MS.