The Kinesin superfamily proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1Balpha is a monomeric motor that conveys mitochondria and plays an important role in cellular function. Here, we used the yeast two-hybrid system to identify the proteins that interacts with KIF1Balpha and found a specific interaction with the mammalian LIN-7 (MALS)-3/vertebrate homology of LIN-7 (Veri) and synaptic scaffolding molecule (S-SCAM). MALS-3 protein bound to the tail region of KIF1Balpha but not to other kinesin family members in the yeast two-hybrid assay. The "T-X-V" motif at the C-terminal end of KIF1Balpha is essential for interaction with MALS-3. In addition, this protein showed specific interactions in the Glutathione S-transferase (GST) pull-down assay. An antibody to MALS-3 specifically coimmunoprecipitated KIF1Balpha associated with MALS-3 from mouse brain extracts. These results suggest that MALS-3, as KIF1Balpha receptor, is involved in the KIF1Balpha-mediated transport.
Animals ; Brain ; Glutathione Transferase ; Humans ; Kinesin* ; Mice ; Microtubules ; Mitochondria ; Organelles ; PDZ Domains* ; Two-Hybrid System Techniques

'Divide and conquer' has been the guiding strategy for the study of protein structure and function. Proteins are divided into domains with each domain having a canonical structural definition depending on its type. In this review, we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function; we call these regions 'extensions'. We focus on the highly abundant PDZ (PSD-95, DLG1 and ZO-1) domain. Using bioinformatics, we find that many PDZ domains have potential extensions and we developed an openly-accessible website to display our results ( http://bcz102.ust.hk/pdzex/ ). We propose, using well-studied PDZ domains as illustrative examples, that the roles of PDZ extensions can be classified into at least four categories: 1) protein dynamics-based modulation of target binding affinity, 2) provision of binding sites for macro-molecular assembly, 3) structural integration of multi-domain modules, and 4) expansion of the target ligand-binding pocket. Our review highlights the potential structural and functional importance of domain extensions, highlighting the significance of looking beyond the canonical boundaries of protein domains in general.
Animals ; Binding Sites ; Humans ; Ligands ; PDZ Domains ; Protein Binding ; Protein Multimerization ; Proteins ; chemistry

Syntenin is overexpressed in multiple human cancers and is newly recognized as a novel regulator in melanoma metastasis. It functions as a scaffolding protein, via its two PDZ domains interacting with multiple transmembrane and cytoplasmic partners to regulate many of the major signaling pathways involved in various cellular processes, such as cell surface receptor clustering, protein trafficking, cytoskeleton remodeling, and activation of transcription factor, and results in the increased abilities for tumor cell growth, adhesion, angiogenesis, invasion and metastasis. The present article attempts to review the structure and functions of syntenin by summarizing our current knowledge on the interacting partners and diverse signaling pathways related to syntenin, and highlight the importance of syntenin as a new potential therapeutic target for the aggressive human melanoma.
Animals ; Biomarkers, Tumor ; genetics ; metabolism ; Humans ; Melanoma ; genetics ; metabolism ; pathology ; Neoplasm Metastasis ; PDZ Domains ; genetics ; Signal Transduction ; Syntenins ; chemistry ; genetics ; metabolism

Syntenin is an adaptor molecule containing 2 PDZ domains which mediate molecular interactions with diverse integral or cytoplasmic proteins. Most of the results on the biological function of syntenin were obtained from studies with malignant cells, necessitating exploration into the role of syntenin in normal cells. To understand its role in normal cells, we investigated expression and function of syntenin in human lymphoid tissue and cells in situ and in vitro. Syntenin expression was denser in the germinal center than in the extrafollicular area. Inside the germinal center, syntenin expression was obvious in follicular dendritic cells (FDCs). Flow cytometric analysis with isolated cells confirmed a weak expression of syntenin in T and B cells and a strong expression in FDCs. In FDC-like cells, HK cells, most syntenin proteins were found in the cytoplasm compared to weak expression in the nucleus. To study the function of syntenin in FDC, we examined its role in the focal adhesion of HK cells by depleting syntenin by siRNA technology. Knockdown of syntenin markedly impaired focal adhesion kinase phosphorylation in HK cells. These results suggest that syntenin may play an important role in normal physiology as well as in cancer pathology.
B-Lymphocytes ; Cytoplasm ; Dendritic Cells, Follicular* ; Focal Adhesion Protein-Tyrosine Kinases* ; Focal Adhesions* ; Germinal Center ; Humans* ; Lymphoid Tissue ; PDZ Domains ; Phosphorylation ; Proteins ; RNA, Small Interfering ; Syntenins*

OBJECTIVETo investigate the role of 83 site in interaction of GluR2 C-terminal and PICK1 PDZ domain.

METHODSDocking structure of PICK1 PDZ domain with GluR2 C terminal PDZ binding motif was built with computer software. After K83 site was substituted by other amino acid, the structure and binding energy were recalculated; meanwhile, site specific mutants were constructed using wild type full length cDNA as template. Mutants were co-transfected with GluR2 into HEK293T cells. After staining, the distribution of PICK1 and GluR2 were observed under confocal microscope.

RESULTSWild type PICK1 and GluR2 formed many co-clusters in HEK293T cells as reported by other research groups; but different K83 mutant had different distribution in HEK293T cells.

CONCLUSIONThe K83 site in PDZ domain of PICK1 is important for the interaction between PICK1 and GluR2. Altering lysine will probably change the hydrophobic interactions, the hydrogen bonds or the electrostatic interactions formed between PICK1 PDZ domain and GluR2 C terminal; accordingly, that will change the binding capacity between PICK1 and GluR2 in varying degrees.

Binding Sites ; Carrier Proteins ; chemistry ; metabolism ; Computer Simulation ; HEK293 Cells ; Humans ; Nuclear Proteins ; chemistry ; metabolism ; PDZ Domains ; Protein Binding ; Receptors, AMPA ; metabolism

OBJECTIVETo identify the binding proteins to PDZ domain of ERBIN.

METHODSUsing PDZ domain of ERBIN as the bait, yeast two-hybrid technology was employed to screen the human lymphocyte leukemia cells MATCHMAKER cDNA library. The protein interaction was identified by immunoprecipitation.

RESULTSA PDZ-binding protein, TAX1, was identified.

CONCLUSIONTAX1 is a novel binding protein to PDZ domain of ERBIN.

Adaptor Proteins, Signal Transducing ; metabolism ; Cell Line, Tumor ; Humans ; Immunoprecipitation ; Intracellular Signaling Peptides and Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; PDZ Domains ; Protein Binding ; Two-Hybrid System Techniques

To detect the effect of PDZ1, domain of postsynaptic density 95 (PSD-95), on apoptosis of hippocampal neurons induced by oxygen-glucose deprivation (OGD), Sprague-Dawley rat hippocampal neurons were infected with PDZ1-viruses after 21 days of plating. Twenty-four hours after infection, cells were treated with OGD for 1.5 h, then were incubated with DAPI and apoptosis-like cells were characterized, or were collected for co-immunoprecipitation and Western blot analyses. The results showed that: (1) PDZ1 overexpression was observed in hippocampal neurons; (2) Apoptosis induced by OGD was obviously decreased in neurons overexpressing PDZ1 (P<0.05); (3) Overexpression of PDZ1 prevented the binding of GluR6 to PSD-95; (4) Overexpression of PDZ1 inhibited MLK3 and JNK1/2 activation induced by OGD. These results indicate that overexpression of PDZ1 may prevent hippocampal neurons from apoptosis induced by OGD.
Animals ; Apoptosis ; Cells, Cultured ; Culture Media ; chemistry ; Disks Large Homolog 4 Protein ; Glucose ; chemistry ; Hippocampus ; cytology ; Intracellular Signaling Peptides and Proteins ; metabolism ; Membrane Proteins ; metabolism ; Neurons ; cytology ; pathology ; Oxygen ; chemistry ; PDZ Domains ; Rats ; Rats, Sprague-Dawley

The control of organ size growth is one of the most fundamental aspects of life. In the past two decades, a highly conserved Hippo signaling pathway has been identified as a key molecular mechanism for governing organ size regulation. In the middle of this pathway is a kinase cascade that negatively regulates the downstream component Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ)/Yorkie through phosphorylation. Phosphorylation of YAP/TAZ/Yorkie promotes its cytoplasmic localization, leads to cell apoptosis and restricts organ size overgrowth. When the Hippo pathway is inactivated, YAP/TAZ/Yorkie translocates into the nucleus to bind to the transcription enhancer factor (TEAD/TEF) family of transcriptional factors to promote cell growth and proliferation. In this review, we will focus on the structural and functional studies on the downstream transcription factor TEAD and its coactivator YAP.
Animals ; Apoptosis ; Cell Proliferation ; Drosophila ; genetics ; metabolism ; Drosophila Proteins ; genetics ; metabolism ; Gene Expression Regulation, Developmental ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Mice ; Nuclear Proteins ; genetics ; metabolism ; Organ Size ; physiology ; PDZ Domains ; Phosphorylation ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Signal Transduction ; physiology ; Trans-Activators ; genetics ; metabolism

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.
Animals ; Brain ; metabolism ; Calcium ; metabolism ; Cells, Cultured ; Cerebellum ; cytology ; Fluorescence Resonance Energy Transfer ; HEK293 Cells ; Humans ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; PDZ Domains ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; Protein Interaction Maps ; Protein Isoforms ; metabolism ; Rats ; Rats, Sprague-Dawley