Gα13 overexpression induces serum response element genes: membrane delocalization and activation state

Gray Robinson McDiarmid

Abstract


Heterotrimeric G proteins consist of α, β, and γ subunits, and activating, GTPase-deficient mutations in the α subunit of several classes have been implicated in tumor progression.  The G12/13 class of α subunits, comprising Gα12 and Gα13, has the unique ability to stimulate oncogenic transformation of cultured cells though overexpression of the wildtype α subunit. However, the mechanism that allows this signaling property of overexpressed wildtype Gα12 or Gα13 is not understood, as G-proteins are not thought to signal outside of the GTP-bound active state. Using transiently transfected HEK293 cells, we examined wildtype forms of Gα13 for ability to stimulate serum response factor (SRF) mediated transcription of genes harboring the serum response element (SRE) promotor, a cell growth signaling mechanism implicated in melanoma and other cancers.  Constitutively active Gα13 showed an approximate 100-fold stimulation of SRE, and unexpectedly, we observed a response fully half this magnitude using wildtype Gα13.  In cell fractionation experiments using an epitope-tagged wildtype Gα13, this transiently expressed protein began appearing in the soluble fraction as its levels increased, whereas endogenous Gα13 remained in the membrane-associated fraction. Under conditions in which Gα13 redistribution was observed, SRE activation showed a sharp increase. This stimulatory effect of wildtype Gα13 was blunted by co-expression of Gβ1 and Gγ2 subunits, suggesting overexpressed Gα13 drives SRE due to stoichiometric imbalance in the heterotrimer. Gβ1/Gγ2 co-expression also changed the subcellular distribution of wildtype Gα13 to a predominantly membrane-associated state.  To examine the activation state within these fractions, we examined the ability of Gα13 to resist trypsin degradation, a feature of GTP-bound Gα subunits. Surprisingly, overexpressed wildtype Gα13 was degraded as fully as mutationally inactivated Gα13, suggesting overexpressed Gα13 that is membrane dissociated does not require activation to signal to SRE and promote cell growth.


Keywords


Cancer; G protein; Signaling

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