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Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER–mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER–mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4. [ABSTRACT FROM AUTHOR]

Prickle is known to be involved in planar cell polarity, including convergent extension and cell migration; however, the detailed mechanism by which Prickle regulates cellular functions is not well understood. Here, we show that Prickle1 regulates front-rear polarization and migration of gastric cancer MKN1 cells. Prickle1 preferentially accumulated at the cell retraction site in close proximity to paxillin at focal adhesions. Prickle1 dynamics correlated with those of paxillin during focal adhesion disassembly. Furthermore, Prickle1 was required for focal adhesion disassembly. CLASPs (of which there are two isoforms, CLASP1 and CLASP2, in mammals) and LL5β (also known as PHLDB2) have been reported to form a complex at cell edges and to control microtubule-dependent focal adhesion disassembly. Prickle1 was associated with CLASPs and LL5β, and was required for the LL5β-dependent accumulation of CLASPs at the cell edge. Knockdown of CLASPs and LL5β suppressed Prickle1- dependent cell polarization and migration. Prickle1 localized to the membrane through its farnesyl moiety, and the membrane localization was necessary for Prickle1 to regulate migration, to bind to CLASPs and LL5β, and to promote microtubule targeting of focal adhesions. Taken together, these results suggest that Prickle1 promotes focal adhesion disassembly during the retraction processes of cell polarization and migration. [ABSTRACT FROM AUTHOR]

Growth factor signaling is involved in the development of various organs, but how signaling regulates organ morphogenesis and differentiation in a coordinated manner remains to be clarified. Here, we show how WNT signaling controls epithelial morphogenetic changes and differentiation using the salivary gland as a model. Experiments using genetically manipulated mice and organ cultures revealed that WNT signaling at an early stage (E12-E15) of submandibular salivary gland (SMG) development inhibits end bud morphogenesis and differentiation into proacini by suppressing Kit expression through the upregulation of the transcription factor MYB, and concomitantly increasing the expression of distal progenitor markers. In addition, WNT signaling at the early stage of SMG development promoted end bud cell proliferation, leading to duct formation. WNT signaling reduction at a late stage (E16-E18) of SMG development promoted end bud maturation and suppressed duct formation. Thus, WNT signaling controls the timing of SMG organogenesis by keeping end bud cells in an undifferentiated bipotent state. [ABSTRACT FROM AUTHOR]

Glypicans are members of the heparan sulfate proteoglycans (HSPGs) and are involved in various growth factor signaling mechanisms. Although HSPGs affect the β-catenin-dependent and -independent pathways of Wnt signaling, how they regulate distinct Wnt pathways is not clear. It has been suggested that the β-catenin-dependent pathway is initiated through receptor endocytosis in lipid raft microdomains and the independent pathway is activated through receptor endocytosis in non-lipid raft microdomains. Here, evidence is presented that glypican-4 (GPC4) is localized to both membrane microdomains and that the localization affects its ability to regulate distinct Wnt pathways. GPC4 bound to Wnt3a and Wnt5a, which activate the β-catenin-dependent and -independent pathways, respectively, and colocalized with Wnts on the cell surface. LRP6, one of Wnt3a coreceptors, was present in lipid raft microdomains, whereas Ror2, one of Wnt5a coreceptors, was localized to non-lipid raft microdomains. Expression of GPC4 enhanced the Wnt3adependent β-catenin pathway and the Wnt5a-dependent β-catenin-independent pathway, and knockdown of GPC4 suppressed both pathways. A GPC4 mutant that was localized to only non-lipid raft microdomains inhibited the β-catenin-dependent pathway but enhanced the β-catenin-independent pathway. These results suggest that GPC4 concentrates Wnt3a and Wnt5a to the vicinity of their specific receptors in different membrane microdomains, thereby regulating distinct Wnt signaling. [ABSTRACT FROM AUTHOR]