Assembly of E-cadherin–based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIγ also interacts with the μ subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIγ or disruption of PIPKIγ binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIγ binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIγ serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate.

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIγ661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIγ661 and Src. The phosphorylation of Y644 results in an ∼15-fold increase in binding affinity to the talin head domain and blocks β-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a “molecular switch” for talin binding between PIPKIγ661 and β-integrin that may regulate dynamic FA turnover.

Peptide amphiphile (PA) nanofibers formed by self-assembly can be customized for specific applications in regenerative medicine through the use of molecules that display bioactive signals on their surfaces. We report here on the use of PA nanofibers with binding affinity for the bone promoting growth factor BMP-2 to create a gel scaffold for osteogenesis. With the objective of reducing the amount of BMP-2 used clinically for successful arthrodesis in the spine, we used amounts of growth factor incorporated in the scaffolds that are 10 to 100 times lower than that those used clinically in collagen scaffolds. The efficacy of the bioactive PA system to promote BMP-2-induced osteogenesis in vivo was investigated in a rat posterolateral lumbar intertransverse spinal fusion model. PA nanofiber gels displaying BMP-2-binding segments exhibited superior spinal fusion rates relative to controls, effectively decreasing the required therapeutic dose of BMP-2 by ten-fold. Interestingly, a 42% fusion rate was observed for gels containing the bioactive nanofibers without the use of exogenous BMP-2, suggesting the ability of the nanofiber to recruit endogenous growth factor. Results obtained here demonstrate that bioactive biomaterials with capacity to bind specific growth factors by design are great targets for regenerative medicine.

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P 2 is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the 2-subunit of the AP2 complex and PIPKI␥661 via a yeast two-hybrid screen. This interaction was confirmed using both the 2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKI␥661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKI␥661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKI␥661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKI␥661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKI␥661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P 2 . RNA interference-based knockdown of PIPKI␥661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKI␥661 in the AP2-mediated endocytosis of transferrin.

The existence of specialized molecular chaperones that interact directly with ribosomes is well established in microorganisms. Such proteins bind polypeptides exiting the ribosomal tunnel and provide a physical link between translation and protein folding. We report that ribosome-associated molecular chaperones have been maintained throughout eukaryotic evolution, as illustrated by Mpp11, the human ortholog of the yeast ribosome-associated J protein Zuo. When expressed in yeast, Mpp11 partially substituted for Zuo by partnering with the multipurpose Hsp70 Ssa, the homolog of mammalian Hsc70. We propose that in metazoans, ribosome-associated Mpp11 recruits the multifunctional soluble Hsc70 to nascent polypeptide chains as they exit the ribosome.