N-Myristoylation is essential for protein phosphatases PPM1A and PPM1B to dephosphorylate their physiological substrates in cells
ABSTRACT Metal-dependent protein phosphatase (PPM, formerly called PP2C) family members play essential roles in regulating a variety of signaling pathways. While searching for protein phosphatase(s) that act on AMP-activated protein kinase (AMPK), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the a subunit of AMPK (AMPKa) in cells. N-myristoylation was also required for two other functions of PPM1A and PPM1B in cells. Although a non-myristoylated mutation (G2A) of PPM1A and PPM1B prevented membrane association, this relocalization did not likely cause the decreased activity toward AMPKa. In in vitro experiments, the G2A mutants exhibited reduced activities toward AMPKa but much higher specific activity against an artificial substrate, pNPP, compared to the wild-type counterparts. Taken together, these observations suggest that N-myristoylation of PPM1A and PPM1B plays a key role in recognition of their physiological substrates in cells.
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- "Lipid modifications of proteins play many roles inside and outside the cell . They facilitate membrane attachment of soluble proteins by enhancing interactions of proteins with either organelle or plasma membranes, occur in signaling and subcellular targeting by directing proteins to various cellular membranes, promote intra-and intermolecular protein–protein interactions, and may modulate protein structure and function      . "
ABSTRACT: VIsinin-LIke Proteins (VILIPs) are a subfamily of the Neuronal Calcium Sensor (NCS) proteins, which possess both N-myristoylation and EF-hand motifs allowing for a putative 'calcium-myristoyl switch' regulation mechanism. It has previously been established that myristoyl conjugation increases the affinity of proteins for membranes, but, in many cases, a second feature such as a cluster of positively-charged residues is needed for stable membrane binding. The interaction of two members of this family, VILIP-1 and VILIP-3, with Langmuir monolayers as membrane models has been investigated in order to study the effects of both myristoylation and the highly basic region containing conserved poly-lysine residues on membrane association kinetics and binding properties. Results show that in the presence of calcium, N-myristoylation significantly increases the kinetic rate of VILIP adsorption to the membrane. Additionally, the proteins bind to negatively charged phospholipids independently of the conjugated myristate moiety. Besides the regulatory effect of calcium on the rate of binding presumably due to exposure of the myristoyl moiety ascribed to their putative 'calcium-myristoyl switch', VILIP-1 and -3 also engage specific interactions with biomimetic membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2). The presence of PIP2 increases the membrane association rates of both VILIPs. Taken together, these results show the major kinetic role of N-myristoylation for membrane binding, and highlight the critical role of specific phosphoinositide interactions for membrane association of members of the VILIP family.Biochimica et Biophysica Acta (BBA) - Biomembranes 07/2014; 1838(10). DOI:10.1016/j.bbamem.2014.06.021 · 3.43 Impact Factor
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ABSTRACT: Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) have been extensively studied in mammals, whereas fungus CaMKs still remain largely uncharacterized. We previously obtained CaMK homolog in Coprinopsis cinerea, designated CoPK12, and revealed its unique catalytic properties in comparison with the mammalian CaMKs. To further clarify the regulatory mechanisms of CoPK12, we investigated post-translational modification and subcellular localization of CoPK12 in this study. In C. cinerea, full-length CoPK12 (65 kDa) was fractionated in the membrane fraction, while the catalytically active fragment (46 kDa) of CoPK12 was solely detected in the soluble fraction by differential centrifugation. Expressed CoPK12-GFP was localized on the cytoplasmic and vacuolar membranes as visualized by green fluorescence in yeast cells. In vitro N-myristoylation assay revealed that CoPK12 is N-myristoylated at Gly-2 in the N-terminal position. Furthermore, calmodulin could bind not only to CaM-binding domain but also to the N-terminal myristoyl moiety of CoPK12. These results, taken together, suggest that the cellular localization and function of CoPK12 are regulated by protein N-myristoylation and limited proteolysis.Journal of Biochemistry 03/2014; DOI:10.1093/jb/mvu018 · 3.07 Impact Factor
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ABSTRACT: The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.PLoS Genetics 05/2014; 10(5):e1004297. DOI:10.1371/journal.pgen.1004297 · 8.17 Impact Factor