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Titolo:
Determination of interaction sites of phospholipase D-1 for RhoA
Autore:
Cai, SM; Exton, JH;
Indirizzi:
Vanderbilt Univ, Sch Med, Dept Mol Physiol & Biophys, Howard Hughes Med Inst, Nashville, TN 37232 USA Vanderbilt Univ Nashville TN USA 37232 Med Inst, Nashville, TN 37232 USA
Titolo Testata:
BIOCHEMICAL JOURNAL
, volume: 355, anno: 2001,
parte:, 3
pagine: 779 - 785
SICI:
0264-6021(20010501)355:<779:DOISOP>2.0.ZU;2-9
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROTEIN-KINASE-C; ADP-RIBOSYLATION FACTOR; RAT-BRAIN; CELLULAR-TRANSFORMATION; TERMINAL DOMAINS; TARGET PROTEINS; EFFECTOR REGION; BINDING DOMAIN; INSERT REGION; GENE FAMILY;
Keywords:
activation; binding; mutation; protein kinase C;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
40
Recensione:
Indirizzi per estratti:
Indirizzo: Exton, JH Vanderbilt Univ, Sch Med, Dept Mol Physiol & Biophys, Howard Hughes Med Inst, 221 Kirkland Hall, Nashville, TN 37232 USA Vanderbilt Univ 221 Kirkland Hall Nashville TN USA 37232 232 USA
Citazione:
S.M. Cai e J.H. Exton, "Determination of interaction sites of phospholipase D-1 for RhoA", BIOCHEM J, 355, 2001, pp. 779-785

Abstract

Phospholipase D (PLD) is regulated by many factors! including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the interaction site of human PLD1 forRhoA is located in its C-terminus, but the exact locus has not been determined. The purpose of the present study was to determine the interaction site of rat PLD1 (rPLD(1)) with RhoA. Selection with phage display of different peptides of rPLD(1) confirmed that GTP-bound RhoA interacted with a site in the amino acid sequence 873-1024 at the C-terminus of rPLD(1). RhoA alsoassociated with this peptide in a GTP-dependent manner in COS-7 cell lysates and the peptide inhibited RhoA stimulation of PLD activity in membranes from COS-7 cells expressing rPLD(1). A series of alanine mutations of non-conserved residues were made in this sequence, and the enzymes were expressed in COS-7 cells and checked for responses to activation of PKC, which interacts with the N-terminus of PLD1, and also to the constitutively active V14RhoA. Mutations in the C-terminus of rPLD(1) (K946A, V950A, R955A and K962A) caused partial loss of V14RhoA stimulation, and double mutations (K946A/K962A, K946A/V950A and K962A/V950A) caused an almost total loss. Co-immunoprecipitation studies also showed that the mutated forms of rPLD(1) described above failed to bind V14RhoA compared with wild-type rPLD(1). whereas rPLD(1) with mutations outside the region K946-K962 bound V14RhoA normally. Itis concluded that basic amino acids in a restricted C-terminal region of rPLD(1) are important for binding of RhoA and its activation of PLD activity.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 09/07/20 alle ore 23:13:34