γ-Synuclein interacts with phospholipase Cβ2 to modulate G protein activation.
Phospholipase Cβ2 (PLC β2) is activated by G proteins and generates calcium signals in cells. PLCβ2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCβ2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCβ2 are associated. In solution, purified γ-synuclein binds to PLCβ2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCβ2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gβγ). Binding of γ-synuclein reduces the catalytic activity of PLCβ2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCβ2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCβ2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gβγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCβ2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCβ2.
Department of Physiology & Biophysics, Stony Brook University, Stony Brook, New York, United States of America.
Urszula
U
Golebiewska
Yuanjian
Y
Guo
Narindra
N
Khalikaprasad
Cassandra
C
Zurawsky
V Siddhartha
VS
Yerramilli
Suzanne
S
Scarlata
D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism*; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D054799:Phospholipase C beta / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051845:gamma-Synuclein / Q000378:metabolism*
D051845:gamma-Synuclein; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins