Volume 8 issue 9 September 2007
A Cytoplasmic PPPSP Motif Determines Megalin´s phosphorylation  and regulates receptor´s  recycling and Surface expression
MI Yuseff , P Farfan, G Bu and MP Marzolo

Supplementary Figure 1. (A) In vitro phosphorylation of the megalin cytoplasmic domain. The GST-MegT fusion protein was phosphorylated in vitro using 100 ng of the catalytic subunit for PKA or PKC in the presence of [γ-³²P] ATP. The reactions incubated with different inhibitors for PKA (PKI) or for PKC (R24571 and GF109293X) are marked (+). The in vitro phosphorylation of megalin by CK-II was assessed by incubating GST alone (negative control) or the GST-MegT fusion protein with 100 ng of the catalytic subunit for CK-II in the presence of [γ-³²P] ATP. Phosphorylation of casein was used as positive control in order to test CK-II activity. The TCA-precipitated proteins were analyzed by 12% SDS-PAGE. (B) Phosphorylation levels of megalin tail minireceptors (mLRP4MegT) containing single point mutations in key residues within PKC or PKA phosphorylation sites and also the minireceptor with the four putative PKC sites and the single PKA site mutated simultaneously (PKC/PKA mut). CHO cell lines expressing these constructs were metabolically labeled with [³⁵S] cysteine or with [³²P] orthophosphate and the minireceptors were immunoprecipitated with an anti-HA antibody. The phosphorylation levels of the single PKA or PKC mutants and of the PKC/PKA mutant were not significantly decreased compared to the wild type receptor, suggesting that these motifs do not constitute important phosphorylation targets in vivo.

Figure 1 (.gif)

Supplementary Figure 2.Effect of protein kinase inhibitors on the megalin tail phosphorylation mutant. (A) CHO cells expressing the mLRP4MegT-PPPAP construct were labeled with [³⁵S] cysteine or with [³²P] orthophosphate in the presence or absence of PKC inhibitors (10 µM of myristoylated peptide and/or 0.5 µM of GF109293X), CK-II inhibitors (DRB and A3 100 µM). (B) The phosphorylation levels were quantified as previously described showing that the residual levels of phosphorylation observed for the PPPAP mutant are dependent on PKC activity.

Figure 2 (.gif)