Association of activated Gαq to the tumor suppressor Fhit is enhanced by phospholipase Cβ

Abstract

Background: G proteins are known to modulate various growth signals and are implicated in the regulation of tumorigenesis. The tumor suppressor Fhit is a newly identified interaction partner of Gq proteins that typically stimulate the phospholipase C pathway. Activated Gαq subunits have been shown to interact directly with Fhit, up-regulate Fhit expression and enhance its suppressive effect on cell growth and migration. Other signaling molecules may be involved in modulating Gαq/Fhit interaction.

Methods: To test the relationship of PLCβ with the interaction between Gαq and Fhit, co-immunoprecipication assay was performed on HEK293 cells co-transfected with different combinations of Flag-Fhit, Gα16, Gα16QL, pcDNA3 vector, and PLCβ isoforms. Possible associations of Fhit with other effectors of Gαq were also demonstrated by co-immunoprecipitation. The regions of Gαq for Fhit interaction and PLCβ stimulation were further evaluated by inositol phosphates accumulation assay using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz.

Results: PLCβ1, 2 and 3 interacted with Fhit regardless of the expression of Gαq. Expression of PLCβ increased the affinities of Fhit for both wild-type and activated Gαq. Swapping of the Fhit-interacting α2-β4 region of Gαq with Gαi eliminated the association of Gαq with Fhit without affecting the ability of the mutant to stimulate PLCβ. Other effectors of Gαq including RGS2 and p63RhoGEF were unable to interact with Fhit.

Conclusions: PLCβ may participate in the regulation of Fhit by Gq in a unique way. PLCβ interacts with Fhit and increases the interaction between Gαq and Fhit. The Gαq/PLCβ/Fhit complex formation points to a novel signaling pathway that may negatively regulate tumor cell growth.

Fig. 1

PLCβs enhance the interaction between Fhit and Gα_q. HEK293 cells were co-transfected with different combinations of Flag-Fhit, Gα₁₆, Gα₁₆QL, pcDNA3 vector, PLCβ1, PLCβ2 or PLCβ3. One day after transfection, cell lysates were prepared and immunoprecipitated with anti-Flag affinity gel. PLCβ1, 2, 3, Gα₁₆, Fhit and α-tubulin of the co-immunoprecipitation (IP) assay and total cell lysate (TCL) were determined by Western blotting

Fig. 2

Fhit interacts with PLCβs. a HEK293 cells were transfected with pcDNA3 vector, PLCβ1, PLCβ2 or PLCβ3 in combination with Flag-Fhit (F) or pFlag-CMV2 (V) vector. Following expression for 1 day, cells were lysed and subjected to co-immunoprecipitation assay with anti-Flag affinity gel. The levels of Fhit and PLCβs were examined by Western blotting. b In the co-immunoprecipitation assay in a, a longer exposure of the anti-PLCβ1 blot showed that endogenous PLCβ1 (indicated by a horizontal arrow) was pulled down by Fhit (indicated by vertical arrows)

Fig. 3

Fhit does not interact with RGS2 or p63RhoGEF. a HEK293 cells were transfected with pcDNA3 vector or HA-tagged RGS2 in combination with pFlag-CMV2 (V) or Flag-Fhit (F). Cell lysates were subjected to co-immunoprecipitation assay with anti-Flag or anti-HA affinity gel. RGS2, Gα_q, Fhit and α-tubulin were detected by Western blotting. b HEK293 cells were transfected with different combinations of Flag-Fhit, myc-p63RhoGEF and Gα_q or Gα_qRC. After 1 day, cells were subjected to the co-immunoprecipation with anti-myc affinity gel. The immunoprecipitates and total cell lysates were analyzed by Western blot

Fig. 4

Fhit does not enhance the association of Gα₁₆QL with PLCβ3. Gα₁₆ or Gα₁₆QL was co-transfected into HEK293 cells with pFlag-CMV2 (Vector) or Flag-Fhit in combination with PLCβ3. One day after transfection, cell lysates were immunoprecipitated with anti-PLCβ3 antibody and Protein G agrose, and subjected to Western blot analysis

Fig. 5

The chimera zα2β4 stimulates PLCβ but does not interact with Fhit. a Schematic representation of the zα2β4 and 16α2β4 chimeras. The linearized secondary structure of Gα_q (filled with white) includes a helical domain (helices A-G) and a GTPase domain (helices 1–5 and strands 1–6). In the secondary structures of Gα₁₆, Gα_z, C128, zα2β4 or 16α2β4, the sequences from Gα₁₆ are filled with black and those from Gα_z are filled with gray. b Inositol phosphates accumulation assays were performed in COS-7 cells transfected with the wild-type or constitutively active mutants of Gα₁₆, Gα_z, C128, zα2β4 or 16α2β4. The relative IP₃ production was quantified. The expressions of the chimeras were examined by the Western blot. * Gα₁₆QL and zα2β4QL significantly increased the IP₃ production (Dunnett’s t test, P < 0.05). c HEK293 cells were transiently co-transfected with Flag tagged Fhit and the wild-type or constitutively active mutants of Gα₁₆, Gα_z, zα2β4 or 16α2β4. Cell lysates were immunoprecipitated with anti-Flag agarose affinity gel (upper panels). Expression levels of Gα₁₆, Gα_z, Flag-Fhit and α-tubulin in the total cell lysate were detected by western blotting (lower panels)

Fig. 6

The binding regions of Fhit and PLCβ on activated Gα_q surface. Molecular surface of activated Gα_q was modeled based on the crystal structures of activated Gα_q and PLCβ3 (PDB: 4GNK). a The location of the Fhit-interacting α2-β4 regions (Gly208-Asp243, purple) relative to the other domains (white) on Gα_q is illustrated. b The contact interface of activated Gα_q with PLCβ3 is highlighted in yellow. c Overlapped binding regions of PLCβ and Fhit on activated Gα_q is shown in green. d PLCβ increases the interaction between Gα_q and Fhit. Gα_q does not enhance the interaction between PLCβ and Fhit. And Fhit is unable to strengthen the association of Gα_q and PLCβ