Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C δ1 and p130

Semmelweis University, Budapeŝto, Budapest, Hungary
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2002; 277(30):27412-22. DOI: 10.1074/jbc.M109672200
Source: PubMed

ABSTRACT The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

Download full-text


Available from: Gyorgy Hajnoczky, Sep 25, 2015
27 Reads
  • Source
    • "Plasma membrane localization of CFP-PH was monitored by TIRF microscopy. Indeed, 10 µM ionomycin induced PIP2 depletion as indicated by the translocation of CFP-PH as described [25], [26] (Fig. 3). However, we found that ionomycin concentrations of up to 3 µM did not alter plasma membrane localization of CFP-PH (Fig. 3). "
    [Show abstract] [Hide abstract]
    ABSTRACT: All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression.
    PLoS ONE 12/2013; 8(12):e82290. DOI:10.1371/journal.pone.0082290 · 3.23 Impact Factor
  • Source
    • "Interestingly, our proposed mechanism also explains the experimental observation that the PLC-!1 PH domain exhibits membrane insertion that occurs independent of PIP 2 but is accelerated by its presence [21]. Drastic conformational changes involving the C-terminal half of the protein may also contribute to stable membrane anchoring of the protein [24] [40]. These findings suggest that the ligand stereospecificity of the protein mainly contributes to searching the membrane containing PIP 2 and that stable anchoring to the lipid membrane is mainly achieved by non-specific membrane binding or insertion rather than PIP 2 binding. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP(3)-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP(3) and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP(3)-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.
    Analytical Biochemistry 09/2012; 431(2):106-114. DOI:10.1016/j.ab.2012.09.012 · 2.22 Impact Factor
  • Source
    • "For comparison, we used a similar plasmid containing a nuclear exclusion sequence (NES). The ability of IP3 to bind to the IP3 binding region of the type I IP3R construct had already been demonstrated [21]. Moreover, we had previously shown the efficiency and selectivity of the IP3 vectors to buffer agonist triggering IP3 dependent Ca 2+ signals, either in the nucleus or in the cytosol [14] [22]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: It is well established that inositol 1,4,5-trisphosphate (IP3) dependent Ca(2+) signaling plays a crucial role in cardiomyocyte hypertrophy. However, it is not yet known whether nuclear IP3 represents a Ca(2+) mobilizing pathway involved in this process. The goal of the current work was to investigate the specific role of nuclear IP3 in cardiomyocyte hypertrophic response. In this work, we used an adenovirus construct that selectively buffers IP3 in the nuclear region of neonatal cardiomyocytes. We showed for the first time that nuclear IP3 mediates endothelin-1 (ET-1) induced hypertrophy. We also found that both calcineurin (Cn)/nuclear factor of activated T Cells (NFAT) and histone deacetylase-5 (HDAC5) pathways require nuclear IP3 to mediate pathological cardiomyocyte growth. Additionally, we found that nuclear IP3 buffering inhibited insulin-like growth factor-1 (IGF-1) induced hypertrophy and prevented reexpression of fetal gene program. Together, these results demonstrated that nuclear IP3 is an essential and a conserved signal for both pathological and physiological forms of cardiomyocyte hypertrophy.
    Journal of Molecular and Cellular Cardiology 07/2012; 53(4):475-86. DOI:10.1016/j.yjmcc.2012.06.017 · 4.66 Impact Factor
Show more