Biologie du cancer du sein

Publications

Année de publication : 2003

Thierry Dubois, Eva Zemlickova, Steven Howell, Alastair Aitken (2003 Feb 5)

Centaurin-alpha 1 associates in vitro and in vivo with nucleolin.

Biochemical and biophysical research communications : 502-8 En savoir plus
Résumé

Centaurin-alpha(1) was originally described as a binding partner for phosphoinositides. In spite of the presence of a putative ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) domain, no ARF-GAP activity has been attributed to centaurin-alpha(1) so far. Thus the function of this protein remains to be determined. In order to better understand its intracellular role, we aimed to identify centaurin-alpha(1) partners. Using affinity chromatography followed by mass spectrometry analysis, we identified several potential centaurin-alpha(1) protein partners. Nucleolin, a nucleolar protein involved in ribosome biosynthesis, was the main centaurin-alpha(1) interacting protein. The interaction between centaurin-alpha(1) and nucleolin was confirmed by Western blot analysis and GST pull down assays. Moreover, we have shown that ectopically expressed centaurin-alpha(1) associates in vivo with endogenous nucleolin in human embryonic kidney 293 cells. In addition, the association between nucleolin and centaurin-alpha(1) was disrupted by RNAse treatment, indicating that RNA integrity was necessary for their binding. This suggested that centaurin-alpha(1) was part of a ribonucleoprotein complex.

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Année de publication : 2002

Thierry Dubois, Steven Howell, Eva Zemlickova, Alastair Aitken (2002 Jun 14)

Identification of casein kinase Ialpha interacting protein partners.

FEBS letters : 167-71 En savoir plus
Résumé

Casein kinase Ialpha (CKIalpha) belongs to a family of serine/threonine protein kinases involved in membrane trafficking, RNA processing, mitotic spindle formation and cell cycle progression. In this report, we identified several CKIalpha interacting proteins including RCC1, high mobility group proteins 1 and 2 (HMG1, HMG2), Erf, centaurin-alpha1, synaptotagmin IX and CPI-17 that were isolated from brain as CKIalpha co-purifying proteins. Actin, importin-alpha(1), importin-beta, PP2Ac, centaurin-alpha1, and HMG1 were identified by affinity chromatography using a peptide column comprising residues 214-233 of CKIalpha. We have previously shown that centaurin-alpha1 represents a CKIalpha partner both in vitro and in vivo. The nuclear protein regulator of chromosome condensation 1 (RCC1) is a guanosine nucleotide exchange factor for Ran which is involved in nuclear transport and mitotic spindle formation. Here we show that CKIalpha and RCC1 interact in brain and in cultured cells. However, the interaction does not involve residues 217-233 of CKIalpha which are proposed from X-ray structures to represent an anchoring site for CKI partners. Formation of the RCC1/CKIalpha complex is consistent with the association of the kinase with mitotic spindles. In conclusion, we have identified a number of novel CKIalpha protein partners and their relations to CKI are discussed.

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Thierry Dubois, Preeti Kerai, Michele Learmonth, Andy Cronshaw, Alastair Aitken (2002 Feb 16)

Identification of syntaxin-1A sites of phosphorylation by casein kinase I and casein kinase II.

European journal of biochemistry / FEBS : 909-14 En savoir plus
Résumé

Casein kinases I (CKI) are serine/threonine protein kinases widely expressed in a range of eukaryotes including yeast, mammals and plants. They have been shown to play a role in diverse physiological events including membrane trafficking. CKI alpha is associated with synaptic vesicles and phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated in vitro by CKI on Thr21. Casein kinase II (CKII) has been shown previously to phosphorylate syntaxin-1A in vitro and we have identified Ser14 as the CKII phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter release by forming part of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, we propose that CKI may play a role in synaptic vesicle exocytosis.

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