1 A new phosphatidylinositol 4,5-bisphosphate binding site located

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Transcript of 1 A new phosphatidylinositol 4,5-bisphosphate binding site located

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    A new phosphatidylinositol 4,5-bisphosphate binding site located in the

    C2 domain of protein kinase C.

    Senena Corbalan-Garcia, Josefa Garcia-Garcia, Jose A. Rodrguez-Alfaro and Juan C.


    Dept. de Bioqumica y Biologa Molecular (A). Facultad de Veterinaria, Universidad de

    Murcia, Apdo. 4021, E-30100 Murcia, Spain.

    1Corresponding author:

    Juan C. Gomez-Fernandez

    Phone: 34 968 36 47 66

    Fax: 34 968 36 47 66

    [email protected]

    running title: role of the lysine-rich cluster of the C2 domain of PKC

    Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

    JBC Papers in Press. Published on November 7, 2002 as Manuscript M209385200 by guest on A

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    In view of the interest shown in PtdIns(4,5)P2 as a second messenger, we study

    the activation of Protein Kinase C by this phosphoinositide. Using two double-mutants

    from two different sites located in the C2 domain of PKC, we have determined and

    characterized the PtdIns(4,5)P2 binding site in the protein, which was found to be

    important for its activation. Thus, there are two distinct sites in the C2 domain: the first,

    the lysine-rich cluster located in the 3 and 4 sheets and which activates the enzyme

    through direct binding of PtdIns(4,5)P2, and the second, the already well described site

    formed by the Ca2+ binding region, which also binds phosphatidylserine as a results of

    which the enzyme is activated. The results obtained in this work point to a sequential

    activation model, in which PKC needs Ca2+ before the PtdIns(4,5)P2-dependent

    activation of the enzyme can occur.

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    Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) plays a key role in

    phosphoinositide signaling and regulates a wide range of processes at many subcellular

    sites. It is primarily detected in the plasma membrane but is also found in secretory

    vesicles, lysosomes, in the endoplasmic reticulum, the golgi, and in the nucleus (1-5).

    PtdIns(4,5)P2 can either bind to intracellular proteins and directly modulate their

    subcellular localization and activity, or it can act as a precursor for the generation of

    different second messengers. For example, several families of phospholipase C

    enzymes are responsible for the hydrolysis of PtdIns(4,5)P2 in cells, leading to the

    production of diacylglycerol and inositol-(1,4,5)-trisphosphate (4, 6), which may, in

    turn, lead to the activation of different proteins such as some PKC isotypes.

    Protein kinase C (PKC) comprises a large family of serine/threonine kinases,

    which is activated by many extracellular signals and plays a critical role in many signal-

    transducing pathways in the cell (7-9). Based on their enzymatic properties, the

    mammaliam PKC isotypes have been grouped into smaller subfamilies. The first group,

    which includes the classical isoforms, , I, II and , can be distinguished from the

    other groups because its activity is regulated by diacylglycerol (DAG) and,

    cooperatively, by Ca2+ and acidic phospholipids, particularly phosphatidylserine (PS).

    Members of the second group are the novel mammalian (, , and ) and yeast PKCs

    which are not regulated by Ca2+. The third group comprises the atypical PKC

    isoforms,, and , whose regulation has not been clearly established, although it is

    clear that they are not regulated by DAG or Ca2+ (8, 10).

    In classical PKC isoenzymes, Ca2+-dependent binding to membranes shows a

    high specificity for 1,2-sn-phosphatidyl-L-serine (11-14). Additionally, this group of

    isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and

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    polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds,

    such as arachidonic acid and free fatty acids (17). Furthermore, in vitro experiments

    have demonstrated that the presence of other anionic phospholipids in the vesicles

    decreases the requirement of phosphatidylserine, suggesting that PKC activation, other

    than the classical activation pathway (activation of phospholipase C and production of

    diacylglycerol by hydrolysis of PtdIns(4,5)P2 and increase of intracytosolic Ca2+) could

    take place in vivo (15).

    In view of the interest in PtdIns(4,5)P2 as a second messenger, several studies

    have addressed the activation of PKC isotypes by this class of lipid (11, 18-24).

    However, the results show little consistency as to which of the different PKC isotypes

    are activated or as regards their specificity for the different lipids employed. These

    conflicting results are probably due to the different ways used by investigators to

    activate the enzyme.

    It has been described that the C2 domains of several proteins, such as

    synaptotagmin (25-29) and rabphilin 3A (30), among others, bind PtdIns(4,5)P2 through

    colinear sequences that consist of highly basic aminoacidic residues (3, 25, 26, 30, 31).

    These lysine-rich sequences probably represent the inositol phosphate-binding portions

    of larger phosphoinositide-binding domains. For example, in synaptotagmin, this basic

    sequence is flanked by regions rich in hydrophobic residues that could mediate acyl

    chain interactions (3).

    When we look at the aminoacidic sequence of the PKC-C2 domain, we observe

    that some of the K/R residues described for synaptotagmin are conserved. Previous

    studies in our laboratory have suggested that the highly positive charged 3-4 sheets

    could interact electrostatically with the negatively charged phospholipids located at the

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    membrane surface (32). Whether or not this is significant in the context of the full-

    length protein is still not clear.

    In this paper, we focus on the characterization of the interaction mechanism

    between PKC-C2 domain and PtdIns(4,5)P2 and the consequent enzyme activation.

    For this purpose, we cloned the PKC C2 domain fused to glutathione-S-transferase

    (GST) and full-length PKC fused to a haemagglutinine (HA) tag, which enabled us to

    perform binding and specific activity studies. Site-directed mutagenesis of key residues

    located in two areas of the C2 domain shed light on the interaction of the classical

    isozyme with PtdIns(4,5)P2 and its activation mechanism.

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    1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-

    sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate

    (POPA), and phosphatidylinositol-4,5-bisphosphate (PIP2) were purchased from Avanti

    Polar Lipids Inc.(Birmingham, AL). Phosphatidylinositol-3,4,5-trisphosphate (PIP3)

    and phosphatidylinositol-3-phosphate (PI(3)P) were purchased from Echelon

    Biosciences Inc (Salt Lake City, UT). Phosphatidylinositol (PI) and

    phosphatidylglycerol (PG) were purchased from Lipid Products (Nutfield, Surrey, UK)

    and phorbol 12-myristate 13-acetate (PMA) from Sigma Chemical Co. (Madrid,


    Construction of expression plasmids

    Rat PKC cDNA was a gift from Drs Nishizuka and Ono (Kobe University,

    Kobe, Japan). The cDNA fragment corresponding to residues 158-285 of the PKC-C2

    domain and mutants was amplified using PCR (33). Full-length PKC mutants were

    generated by PCR site-directed mutagenesis (14, 34). All contructs, both wild type and

    mutant genes, were subcloned into the mammalian expression vector pCGN (a gift from

    Dr. Tanaka, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). This vector

    contains the cytomegalovirus promoter and the multicloning sites that allow expression

    of the genes fused 3 to the haemagglutinine (HA) epitope (35). All constructs were

    confirmed by DNA sequencing.

    The cDNA fragment corresponding to residues 158-285 of the PKC-C2

    domain and mutants was amplified using PCR (33).

    Expression and purification of GST-PKC-C2

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    The pGEX-KG plasmid containing the PKC-C2 domain was transformed into

    HB101 E. coli cells. Proteins were expressed and purified as described in a previous

    work (33).

    Cell culture, transfection and purification of PKC

    HEK293 cells were grown in Dulbeccos modified Eagles medium (DMEM)

    with 10% of fetal calf serum (FCS). Transfection was performed with the Ca2+

    phosphate method described by Wigler el al. (36). Protein purification was performed

    as described by Conesa-Zamora et al., (14).

    Phospholipid binding measurements

    a) Standard assay. The procedure described by Davletov and Sdhof (37), was

    used with minor modifications. A total of 10 g of PKC-C2 domain bound to