Fig 2: Afadin-deficient human breast cancer cells are impaired in their ability to metastasize to the liver. (A) A pooled population (n = 3 independent clones) of MDA-MB-231TR cells lacking Afadin (AF6Crispr) or harboring either an empty vector (EV), short Afadin (sAF6), or long Afadin (lAF6) were analyzed by immunoblot analysis, with α-Tubulin serving as a loading control. (B) Tumor growth in the mammary fat pad was measured for the cell populations described in A. (C) Liver metastatic burden (tumor area per tissue area) was analyzed following splenic injection of Afadin-proficient and Afadin-deficient MDA-MB-231TR breast cancer cells. (*) P < 2 × 10−3. (D) Representative images of the cardiac liver lobe for each cell population are shown. Metastatic lesions are outlined by dotted lines. Bar, 2 mm.

Fig 3: Afadin is phosphorylated by S6K1 in Tsc1-mutant conditions.a Cells were serum starved overnight and subsequently amino acid starved with EBSS (Earle’s Balanced Salt Solution) for 1.5 h with or without the indicated treatments. They were then collected and cell lysates were probed with the indicated antibodies. Asterisk represents non-specific cross-reactive bands. b Densitometry analysis and relative quantification of the phosphorylated forms of the indicated proteins. Actin was used as a loading control. Mean ± SEM. n = 4 independent experiments were analyzed. c Western blots of mouse whole-kidney extracts at pre-cystic age (post-natal day 20), randomly fed, and probed with the indicated antibodies. d Densitometry analysis and relative quantification of the phosphorylated form of Afadin. Actin was used as a loading control. Mean ± SEM. n = 3 Tsc1 f/f, n = 3 Tsc1 f/f; S6k1 −/−, n = 4 Ksp-Cre; Tsc1 f/f, and n = 4 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were analyzed. e Western blot of protein lysates extracted from patient samples. Renal tumors (t) or normal tissue adjacent to renal tumor (n) were collected from TSC patients and non-TSC patients. Protein lysates then probed with the indicated antibodies. f Densitometry analysis and fold change of TSC patients over non-TSC patients of the indicated proteins relative to the indicated loading control. Mean ± SEM. n = 5 TSC patients, n = 7 non-TSC patients were analyzed. g Analog-sensitive S6K1 was co-transfected in Tsc1/S6k1 dKO mIMCD3 cells with FLAG-Afadin-WT. An in vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S both in presence and in absence of 20 nM rapamycin. After immunoprecipitation using an anti-FLAG antibody, the thio-phosphorylation of Afadin was revealed by western blot using an anti‐thiophosphate ester antibody. Expression levels of Afadin, phospho-S1795-Afadin, FLAG and phospho-RPS6 were revealed by western blot on total extracts using the indicated antibody. Source data are provided as a Source Data file.

Fig 4: Phosphoproteomic screening of Tsc1 single knockout versus Tsc1/S6k1 double knockout cells.a Volcano plots of AGC kinase-motif antibody enrichment and b IMAC enrichment. The x-axis shows log2-ratio for phosphopeptides between samples and the y-axis shows the −log10 p-value. The horizontal blue bar represents the statistical cut-off of significance (p value of 0.05). Red dots represent downregulated phospho-peptides in the Tsc1/S6k1 double knock-out genotype and green dots represent up-regulated phospho-peptides. Afadin phospho-peptide is represented in yellow. The numbers in the chart represent the number of statistically significant differentially enriched peptides. Representative peptides and their sites of modification are shown. c Gene ontology analysis, using DAVID, showing statistically significant gene set enrichments comparing the IMAC statistically significant differentially phosphorylated proteins versus the entire IMAC enrichment set. Source data are provided as a Source Data file.

Fig 5: Mutation of residues at the interaction interface inhibit dephosphorylation of Afadin by PTPRK.(A) Time course of pNPP dephosphorylation by WT (black) and double mutant (DM, red) PTPRK-ICD. Error bars represent ± SEM of n = 2 independent experiments. (B) Immunoblot analysis of pervanadate-treated MCF10A lysates incubated for 1.5 hr at 4°C with 0.3 µM of PTPRK-ICD WT or DM. (C) Immunoblot analysis of pervanadate-treated MCF10A lysates incubated for 1.5 hr at 4°C with 0.3 µM of PTPRK-ICD WT or DM followed by pTyr immunoprecipitation (IP). Dephosphorylated proteins are depleted from pTyr-IPs and/or enriched in supernatants (sup.). Gels and blots shown in this figure are representative of n = 3 independent experiments. Figure 5—source data 1.Uncropped, unedited blots for Figure 5B. Figure 5—source data 2.Uncropped, unedited blots for Figure 5C.