N-Myristoylation is essential for protein phosphatases PPM1A and PPM1B to dephosphorylate their physiological substrates in cells

Biochemical Journal (Impact Factor: 4.4). 10/2012; 449(3). DOI: 10.1042/BJ20121201
Source: PubMed


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.

  • Source
    • "Lipid modifications of proteins play many roles inside and outside the cell [4]. 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 [5] [6] [7] [8] [9] [10]. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jul 2014 · Biochimica et Biophysica Acta (BBA) - Biomembranes
  • [Show abstract] [Hide abstract]
    ABSTRACT: In a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32. CoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined. CoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase. CoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses. In C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses.
    No preview · Article · Mar 2011 · Biochimica et Biophysica Acta
  • [Show abstract] [Hide abstract]
    ABSTRACT: Multifunctional Ca2+/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.
    No preview · Article · Mar 2014 · Journal of Biochemistry
Show more