Figure 5. RFFL is the E3 ligase for PRR5L.

A) LPA induces Rffl expression dependently of Gα12. WT or Gna12^−/− MEFs were stimulated with LPA, and the relative expression levels of Rffl were determined by qRT-PCR.

B) Gα12, but not Gα13, upregulates RFFL expression. HEK293T cells were transfected with Gα12^QL or Gα13^QL for 24 hrs or treated with LPA (5 μM) for 2 hrs. Relative expression levels of RFFL were determined by qRT-PCR.

C) Knockdown of RFFL abolishes the effects of LPA on PRR5L stability regulation and PKC HM and MARCKS phosphorylation. HEF293T cells were transfected with the RFFL siRNAs for 72 hrs and stimulated with LPA (5 μM, 2hrs) before Western analysis. Normalized quantification of immunoblots was performed from independent experiments. Data are presented as means ± S.D. (*, p<0.01, Student’s t-Test, n=3).

D) RFFL interacts with PRR5L. HEK293T cells were transfected with RFFL-Flag and PRR5L-HA as indicated, and immunoprecipitation was carried out using an anti-Flag antibody 24 hrs after transfection.

E) LPA treatment, expression of RFFL, or Gα12 stimulates endogenous PRR5L polyubiquitination. HEK293T cells were transfected with His-ubiquitin, Gα12^QL, and/or RFFL-FLAG for 24 hrs and pretreated with MG132. Some of the samples were stimulated with LPA (5 μM, 2 hrs). Cell lysates were subjected to Ni-NTA bead pull-down, followed by Western analysis with a PRR5L antibody.

F) RFFL catalyzes PRR5L ubiquitination in vitro.

G) Expression of K48R ubiquitin blocks PRR5L polyubiquitination by RFFL.