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

PLCβ2 and γ-synuclein associate in cells.

<p><b>A </b><b>–</b> Co-immunofluroescence studies showing the colocalization of γ-synuclein (green) and PLCβ2 (red) in two breast cancer cell lines, MCF10A and MDA-MB-231. <b>B-</b> Co-immunoprecipation of endogenous γ-synuclein and PLCβ2 in MDA-MB-231 cells.</p

γ-Synuclein inhibits the enzymatic activity PLCβ2.

<p><i>(left)</i> Change in 20 nM PLCβ2 activity with increasing amounts of γ-synuclein. (<i>right</i>) Ratio of PLCβ2 activity with increasing substrate in the presence and absence of 10 mM Ins(1,4,5)P₃ showing that γ-synuclein promotes product inhibition.</p

γ-Synuclein binding to PLCβ2 results in an apparent increase in G protein activation.

<p><b><i>Top panels</i></b> – Apparent activation of 20 nM PLCß2, as calculated by the ratio of the activity of PLCβ2 complexed with Gαq(<i>left</i>) or Gßγ (<i>right</i>) over PLCβ2 alone, due to reversal of γ-synuclein by G protein subunits. <b><i>Bottom</i></b> – Activity studies showing that Rac1 does not interfere with inhibition of PLCβ2.</p

PLCβ2 and γ-synuclein associate in solution.

<p>Normalized change in fluorescence intensity of 2 nM CPM- PLCβ2in solution as purified γ-synuclein is added where the total intensity increase was 39±4%. The data shown are corrected for dilution and background, which was less than 1% of the signal, and are an average of 3 sets of measurements.</p