Jin Z , Chung JW , Mei W , Strack S , He C , Lau GW , Yang J .

R01 NS043254 NINDS NIH HHS , HL090699 NHLBI NIH HHS , R01NS043254 NINDS NIH HHS , R01 NS056244 NINDS NIH HHS , NS056244 NINDS NIH HHS , R01 GM093217 NIGMS NIH HHS , R01 HL090699 NHLBI NIH HHS , R56 NS056244 NINDS NIH HHS , R01GM093217 NIGMS NIH HHS

	FIGURE 1:. B56-containing PP2As regulate nuclear localization of Fam13a. (A) Coimmunoprecipitation (CoIP) showing the interaction between FAM13A and B56ε. B56ε-FLAG and myc-FAM13A were expressed in HEK293T cells alone or in combination. Cells were harvested and subjected to CoIP with an anti-FLAG antibody (left) or an anti-myc antibody (right). (B) Western blot showing that FLAG-B56ε coimmunoprecipitated with full-length Fam13a and 1–609 but not other Fam13a deletion constructs (1–513, 1–393, 1–341, 1–158, and ∆513x609). In addition, FLAG-B56ε strongly interacted with 460–609 but only very weakly with 513–609 and 513–693. (C) Immunofluorescence images showing the nuclear localization of Fam13a in control (DMSO) cells and cytoplasmic localization of Fam13a in OA-treated cells. (D) Western blot showing knockdown of endogenous B56ε by a siRNA against B56ε (siB56ε). (E) Immunofluorescence images showing the nuclear localization of Fam13a in control (siGFP) and B56ε knockdown (siB56ε) cells. (F) Immunofluorescence images showing subcellular localization of wild-type Fam13a in vehicle or doxycycline-treated Aα exchange cells. Wild-type Fam13a was nuclear in vehicle-treated DTP177AAA and AαWT cells. Doxycycline treatment induced cytoplasmic sequestration of Fam13a in DTP177AAA cells but not in AαWT cells. Shown in C, E, and F are representative staining patterns. The percentage of cells displaying nuclear (N), cytoplasmic (C), or even distribution between nucleus and cytoplasm (N = C) are graphed. At least 100 cells from each sample were scored in each experiment. (G) Fractionation of control (DMSO-treated) and OA-treated A549 cells. Endogenous Fam13a protein in nuclear and cytosolic fractions was analyzed by Western blot. β-Tubulin and C/EBPβ served as marker for cytosolic and nuclear fractions, respectively.
	FIGURE 2:. Identification of NLSs in Fam13a. (A) Schematic diagram of wild-type Fam13a and NLS mutants (RR340AA, RR531AA, and RR340;531AA). (B) Representative immunofluorescence images showing the nuclear localization of wild-type Fam13a and mislocalization of NLS mutants in NIH3T3 cells. Bar graphs show percentage of cells that exhibit nuclear, cytoplasmic, or homogeneous subcellular distribution. R, arginine; A, alanine.
	FIGURE 3:. Interaction between Fam13a and 14-3-3. (A) GST pull down, showing that Fam13a interacted with bacterially expressed GST–14-3-3η and GST–14-3-3ε but not GST. (B) Western blot, showing coimmunoprecipitation of myc-Fam13a and FLAG–14-3-3η. FLAG–14-3-3η was detected when myc-Fam13a was immunoprecipitated (middle). Conversely, myc-Fam13a was detected when FLAG-14-3-3η was immunoprecipitated (right). (C) GST pull-down assay, showing that GST–14-3-3 interacts with the full-length Fam13a and some Fam13a deletion constructs (1–609, 1–513, 1–393, and 1–341). GST-14-3-3 failed to pull down 1–158. (D) Western blot, showing that truncation of residues 315–329 decreased the binding between GST–14-3-3 and Fam13a.
	FIGURE 4:. The 14-3-3 binding domain is important for cytoplasmic sequestration of Fam13a in response to PP2A inhibition. (A) Representative immunofluorescence images, showing the subcellular distribution of Fam13a and Δ315–329 in control (DMSO) and OA-treated NIH3T3 cells. Fam13a and Δ315–329 were primarily nuclear in control (DMSO) NIH3T3 cells. OA treatment induced cytoplasmic localization of Fam13a. Δ315–329 remained nuclear in the majority of OA-treated cells. (B) Representative immunofluorescence images, showing subcellular distribution of Fam13a and Δ315–329 in control (vehicle-treated) and doxycycline-treated Aα exchange (DTP177AAA) cells. Fam13a and Δ315–329 were nuclear in vehicle-treated cells. After doxycycline treatment, Fam13a was mislocalized. Δ315–329 remained nuclear in the majority of treated cells. Bar graphs in A and B indicate the percentage of cells that exhibit nuclear, cytoplasmic, or homogeneous subcellular distribution. (C) The 14-3-3 binding motif (residues 315–329) is required for the effect of PP2A inhibition on binding between Fam13a and bacterially expressed GST–14-3-3η in a GST pull-down assay. Fam13a- and Δ315–329–expressing cells were treated with DMSO or OA. Cell lysates were used for the GST pull-down assay. Note that OA treatment significantly enhanced interaction between Fam13a and GST–14-3-3. Compared to wild-type Fam13a, Δ315-329 showed much weaker interaction with 14-3-3 after OA treatment. (D) The 14-3-3 binding motif (residues 315–329) is required for the effect of B56s knockdown on binding between Fam13a and GST–14-3-3 in a GST pull-down assay. Fam13a- and Δ315–329–expressing Aα exchange cells were treated with vehicle or doxycycline. Cell lysates were used in a GST pull-down assay. Doxycycline treatment significantly enhanced the interaction between Fam13a and 14-3-3. However, Δ315–329 showed weaker interaction with 14-3-3 compared with wild-type Fam13a after doxycycline treatment.
	FIGURE 5:. Akt promotes cytoplasmic sequestration of Fam13a and binding between Fam13a and 14-3-3. (A) Representative immunofluorescence images, showing the subcellular distribution of Fam13a when cotransfected with an empty vector or Akt-myr in NIH3T3 cells. (B) Representative immunofluorescence images, showing subcellular localization of Fam13a in NIH3T3 cells treated with DMSO (control), OA, wortmannin, LY294002, OA plus wortmannin, or OA plus LY294002. Fam13a localized in the nucleus in control (DMSO) cells. OA-treated cells exhibited cytoplasmic localization of Fam13a. Fam13a remained in the nucleus in wortmannin, LY294002-, OA/wortmannin-, or OA/LY294002-treated cells. Bar graphs in A and B indicate the percentage of cells that exhibit nuclear, cytoplasmic, or homogeneous subcellular distribution. (C) Inhibition of Akt activation by wortmannin reduces OA-induced binding between Fam13a and 14-3-3 in a GST pull-down assay. Fam13a-expressing NIH3T3 cells were treated with DMSO (control), OA, wortmannin, or OA plus wortmannin. Cell lysates were used in a GST pull-down assay. OA treatment strongly enhanced the binding between Fam13a and GST–14-3-3. This effect of OA treatment was reduced by wortmannin treatment. Wortmannin treatment itself had minimal effect on the binding between Fam13a and GST-14-3-3.
	FIGURE 6:. Ser-322 is important for subcellular distribution of Fam13a and its interaction with 14-3-3. (A) Representative immunofluorescence images, showing the subcellular distribution of Fam13a and S322A in NIH3T3 cells treated with DMSO (control) or OA. Bar graph indicates the percentage of cells that exhibit nuclear, cytoplasmic, or homogeneous subcellular distribution. (B) A GST pull down, showing that Ser-322 is important for the binding between Fam13a and 14-3-3 and the effect of OA on this interaction. Fam13a- and S322A-expressing NIH3T3 cells were treated with DMSO (control) or OA. Cell lysates were used in a GST pull-down assay. Interaction between Fam13a and 14-3-3 was markedly enhanced in OA-treated cells. In contrast, S322A mutant showed weaker interaction with 14-3-3 in OA-treated cells. (C) Western blot, showing the specificity of the anti-phosphoS322 (pS322) antibody. Note that the anti-pS322 antibody strongly reacted with Fam13a. It did not react with Δ315–329 but recognized S322A weakly. (D) Western blot, showing Ser-322 phosphorylation in NIH3T3 cells treated with DMSO (control), OA, wortmannin, or OA plus wortmannin. pS322 level was increased by OA treatment. The effect of OA on pS322 was reduced by wortmannin treatment. (E) Western blot, showing that overexpression of B56β, B56γ, B56δ, and B56ε decreased Ser-322 phosphorylation. All B56 expression constructs were FLAG tagged.
	FIGURE 7:. Activation of Wnt signaling by Fam13a. (A) Morphology of uninjected and Fam13a RNA (1 ng)–injected Xenopus embryos at the tadpole stage. Embryos injected with Fam13a developed partial secondary axes (arrowheads). (B) Expression of siamois, Xnr3, chordin, noggin, sizzled, sox17, and Xbra in control and Fam13a-injected embryos. Embryos were harvested at the early gastrula stage. (C) Epistasis analysis showing that the expression of siamois and Xnr3 in Fam13a-overexpressed animal caps was blocked by coexpression of axin or GSK3ß. (D) Western blot showing that overexpression of Fam13a increased the expression of myc–β-catenin in Xenopus embryos. At the two-cell stage, embryos were injected with a mixture of myc–β-catenin (100 pg) and myc-GFP (50 pg) RNAs or a mixture of myc–β-catenin, myc-GFP, and myc-Fam13a (1 ng). Embryos were harvested at stage 9. (E) RT-PCR, showing the expression of siamois and Xnr3 in Fam13a-, ∆315–329–, or RR340;531AA-overexpressed animal caps. As a control, the levels of injected Fam13a, ∆315-329, and RR340;531AA RNAs were monitored. (F) Western blot, showing that overexpression of Fam13a increased the expression of myc–β-catenin in A549 cells. (G) Dual-luciferase assays, showing that overexpression of Fam13a activated TOPFlash and enhanced the activity of β-catenin in A549 cells. FOPFlash, which contains mutations in the TCF binding sites, was included as a negative control. Firefly luciferase activity was normalized to the activity of Renilla luciferase (TK-RL). (H) Overexpression of Fam13a and ∆315–329, but not RR340;531, enhanced the activity of β-catenin in the TOPFlash luciferase assay in A549 cells. Firefly luciferase activity was normalized to the activity of Renilla luciferase. (I) Western blot, showing coimmunoprecipitation of Flag-Fam13a and myc-axin. Of note, we did not detect any interaction between Flag-Fam13a and myc-GSK3β. (J) Western blot showing that overexpression of Fam13a reduced the expression of HA-Axin in A549 cells. The expression level of myc-GSK3ß was not altered by Fam13a overexpression.
	FIGURE 8:. Generation and analysis of Fam13a-knockout mice. (A) RT-PCR, showing the expression of FAM13A in adult mouse tissues. (B) Schematic diagram showing the knockout strategy. Arrowheads indicate primers used for genotyping. KO-F and GT-B were used to detect the knockout allele (KO). WT-F and GT-B were used to detect the wild-type allele (WT). (C) Genotyping PCR showing the genotype of offspring from intercrossing between Fam13a+/− mice. (D) RT-PCR, showing the expression of Fam13a in wild-type, heterozygous, and knockout animals. (E) Sequence of the PCR product amplified from a Fam13a−/−-knockout animal, showing deletion of exon 5. (F) Histological analysis of the large airways (left) and alveoli (right) in a wild-type mouse and a FAM13A-knockout mouse. (G) Representative real-time RT-PCR result, showing the levels of axin2 mRNA in control and Fam13a−/− lungs. (H) Representative real-time RT-PCR result, showing that knockdown of Fam13a reduced the level of axin2 mRNA in A549 cells.
	FIGURE 7:. Activation of Wnt signaling by Fam13a. (A) Morphology of uninjected and Fam13a RNA (1 ng)–injected Xenopus embryos at the tadpole stage. Embryos injected with Fam13a developed partial secondary axes (arrowheads). (B) Expression of siamois, Xnr3, chordin, noggin, sizzled, sox17, and Xbra in control and Fam13a-injected embryos. Embryos were harvested at the early gastrula stage. (C) Epistasis analysis showing that the expression of siamois and Xnr3 in Fam13a-overexpressed animal caps was blocked by coexpression of axin or GSK3ß. (D) Western blot showing that overexpression of Fam13a increased the expression of myc–β-catenin in Xenopus embryos. At the two-cell stage, embryos were injected with a mixture of myc–β-catenin (100 pg) and myc-GFP (50 pg) RNAs or a mixture of myc–β-catenin, myc-GFP, and myc-Fam13a (1 ng). Embryos were harvested at stage 9. (E) RT-PCR, showing the expression of siamois and Xnr3 in Fam13a-, ∆315–329–, or RR340;531AA-overexpressed animal caps. As a control, the levels of injected Fam13a, ∆315-329, and RR340;531AA RNAs were monitored. (F) Western blot, showing that overexpression of Fam13a increased the expression of myc–β-catenin in A549 cells. (G) Dual-luciferase assays, showing that overexpression of Fam13a activated TOPFlash and enhanced the activity of β-catenin in A549 cells. FOPFlash, which contains mutations in the TCF binding sites, was included as a negative control. Firefly luciferase activity was normalized to the activity of Renilla luciferase (TK-RL). (H) Overexpression of Fam13a and ∆315–329, but not RR340;531, enhanced the activity of β-catenin in the TOPFlash luciferase assay in A549 cells. Firefly luciferase activity was normalized to the activity of Renilla luciferase. (I) Western blot, showing coimmunoprecipitation of Flag-Fam13a and myc-axin. Of note, we did not detect any interaction between Flag-Fam13a and myc-GSK3β. (J) Western blot showing that overexpression of Fam13a reduced the expression of HA-Axin in A549 cells. The expression level of myc-GSK3ß was not altered by Fam13a overexpression.

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