The regulation of endocytosis by kinases: cell biology meets genomics

The mechanisms of signal transduction and vesicular transport have traditionally been studied in isolation, but recent studies make it clear that the two processes are inextricably linked. A new genome-wide analysis of human kinases using RNA interference shows an unexpected depth and complexity to the interactions between these processes.     

The interaction of versican with its binding partners

Versican belongs to the family of the large aggregating chondroitin sulfate proteoglycans located primarily within the extracellular matrix （ECM）. Versican, like other members of its family, has unique N- and C-terminal globular regions, each with multiple motifs. A large glycosaminoglycan-binding region lies between them. This review will begin by outlining these structures, in the context of ECM proteoglycans. The diverse binding partners afforded to versican by virtue of its modular design will then be examined. These include ECM components, such as hyaluronan, type Ⅰ collagen, tenascin-R, fibulin-1, and -2, fibrillin-1, fibronectin, P- and L-selectins, and chemokines. Versican also binds to the cell surface proteins CD44, integrin β1, epidermal growth factor receptor, and P-selectin glycoprotein ligand-1. These multiple interactors play important roles in cell behaviour, and the roles of versican in modulating such processes are discussed.     

PKB/Akt partners with Dab2 in albumin endocytosis

Albumin in the glomerular filtrate is normally retrieved by concerted efforts of clathrin, LDL-type receptor megalin- and clathrin-associated sorting proteins. In glomerular diseases, albumin overload triggers a proapoptotic and inflammatory response contributing to tubulointerstitial fibrosis and tubular atrophy. The relationship between albumin overload-induced proximal tubule injury and albumin endocytosis remains to be discovered. We investigated presence of a possible overlap between endocytosis and cell survival. We showed a novel interaction between prosurvival protein, protein kinase B (PKB/Akt), and adaptor protein, disabled 2 (Dab2), with coimmunoprecipitation. Further delineation of this interaction by GST pull-down experiments utilizing different Dab2 constructs identified proline-rich domain as the interacting partner. Expression of Dab2 and PKB/Akt was downregulated at high concentrations of albumin associated with apoptosis. We then examined the physiological relevance of this interaction with functional studies. Overexpression of PKB/Akt increased albumin uptake in human proximal tubule cells. Conversely, inhibition of PKB/Akt with a nonselective Akt/PKB signaling inhibitor-2 and a dominant negative construct of PKB/Akt resulted in a decrease in albumin uptake. Inhibition of Dab2 by silencing RNA abolished PKB/Akt-induced albumin uptake demonstrating the physiological importance of this novel interaction. We concluded that PKB/Akt is part of an endocytic machinery and it mediates albumin uptake through its interaction with Dab2. The role that PKB/Akt plays in the endocytic cascade may dictate its decreased expression in proteinuric states in an attempt to limit albumin endocytosis that may tilt the balance between cell survival and apoptosis toward cell death.     

Cholesterol regulation of rab-mediated sphingolipid endocytosis

Despite a tight regulation of its intracellular content, cholesterol is found accumulated in pathological conditions such as sphingolipidosis as well as after cell treatment with drugs like hydrophobic amines. Furthermore, cellular cholesterol increases when cultured cells approach confluence. Under these conditions, the endocytic pathways of plasma membrane sphingolipids are differently affected. In this short review, we will summarize recent results from our laboratory as well as those of other groups, indicating that the intracellular accumulation of cholesterol inhibits the dissociation of rab GTPases from the target membranes, causing the alteration of rab-mediated membrane traffic.     

Protein kinase C binding partners

Members of the protein kinase C family respond to second messengers and are involved in controlling a broad array of cellular functions. The overlapping specificity and promiscuity of these proteins has promoted the view that specific binding proteins constrain individual family members to create the appropriate specificity of action. It is speculated that such protein kinase C-regulator protein interactions affect substrate availability as well as exposure to allosteric activator(s) and that consequent interactions specify cellular location and impose integration with other signaling systems. These predicted features have been realized in the identification of many protein kinase C interacting proteins and examples of these are discussed.     

Phagocytosis: dynamin's dual role in phagosome biogenesis

Dynamins have a well-established role in the fission of vesicles at sites of endocytosis. In phagocytosis, however, a role for certain dynamin isoforms has been reported in the full extension of pseudopods during phagosome formation, not in fission of the phagocytic vacuole. Recent studies in Caenorhabditis elegans have now uncovered a new function of dynamin in phagosome maturation.     

The quest for ligands and binding partners of atypical cadherin FAT1

A recent study in Scientific Reports identified glypican-3 (GPC3) as a novel extracellular interacting protein for FAT1 in hepato-cellular carcinoma (HCC) cells. FAT1 is a large transmembrane atypical cadherin with limited knowledge existing about its binding partners. While in Drosophila, dachsous (ds), another transmembrane member of the cadherin superfamily, is known to function as FAT1 ligand, no ligand is known in mammals so far. The revelation of GPC3 as a potential binding partner of FAT1 extracellular domain unfolds an opportunity to study potential triggers of FAT1 signaling in cancers. Available inhibitors of GPC3 in various phases of clinical trials also present an attractive option to curb GPC3-FAT1 signaling in tumors that overexpress these proteins.     

Non-carbohydrate binding partners/domains of animal lectins

• 1.1. Protein-carbohydrate interactions are involved in a large number of biologically important recognition processes.
• 2.2. Among the participating classes of proteins lectins are defined as carbohydrate-binding proteins other than an antibody or an enzyme.
• 3.3. In addition to the essential carbohydrate-binding domain other functionally and/or structurally important sites, defined by sequence comparison or by experimental demonstration of protein-protein interactions, can be present within the lectin molecule and may be relevant for its physiological significance.
• 4.4. Sequence motifs of lectins for protein-protein interactions include amino acid structures designed for cell adhesion, growth regulatory biosignalling, intracellular routing and enzymatic activity.
• 5.5. Elucidation of the complete functional role(s) of a lectin requires accurate delineation of its carbohydrate and, if present, of its protein ligands.
• 6.6. Presence of more than one carbohydrate-binding domain in a single lectin, potential ligand properties of the glycopart of a lectin, regulatory interplay between different sites and possible interaction of complementarily shaped peptide sequences to the sugar-recognizing site should all be assessed in the quest to comprehensively explain the physiological role(s) of a lectin.

     

Protein phosphatase-1 binding to scd5p is important for regulation of actin organization and endocytosis in yeast

SCD5, an essential gene, encodes a protein important for endocytosis and actin organization in yeast. Previous two-hybrid screens showed that Scd5p interacts with Glc7p, a yeast Ser/Thr-specific protein phosphatase-1 (PP1) that participates in a variety of cellular processes. PP1 substrate specificity in vivo is regulated by association with different regulatory or targeting subunits, many of which have a consensus PP1-binding site ((V/I)XF, with a basic residue at the -1 or -2 position). Scd5p contains two of these potential PP1-binding motifs: KVDF (amino acids 240-243) and KKVRF (amino acids 272-276). Deletion analysis mapped the PP1-binding domain to a region of Scd5p containing these motifs. Therefore, the consequence of mutating these two potential PP1-binding sites was examined. Although mutation of KVDF had no effect, alteration of KKVRF dramatically reduced Scd5p interaction with Glc7p and resulted in temperature-sensitive growth. Furthermore, this mutation caused defects in fluid phase and receptor-mediated endocytosis and actin organization. Overexpression of GLC7 suppressed the temperature-sensitive growth of the KKVRF mutant and partially rescued the actin organization phenotype. These results provide evidence that Scd5p is a PP1 targeting subunit for regulation of actin organization and endocytosis or that Scd5p is a PP1 substrate, which regulates the function of Scd5p in these processes.     

Cysteine cathepsin non-inhibitory binding partners: modulating intracellular trafficking and function

Cysteine cathepsins play a fundamental role in tumor growth, invasion and migration, angiogenesis, and the metastatic cascade. Evidence of their overexpression in a wide array of human tumors has been well documented. Cysteine cathepsins seem to have a characteristic location-function relationship that leads to non-traditional roles such as those in development and pathology. For example, during tumor development, some cysteine cathepsins are found not just within lysosomes, but are also redistributed into presumptive exocytic vesicles at the cell periphery, resulting in their secretion. This altered localization contributes to non-lysosomal functions that have been linked to malignant progression. Mechanisms for altered localization are not well understood, but do include the interaction of cysteine cathepsins with binding partners that modulate intracellular trafficking and association with specific regions on the cell surface.     

Formation of the gap junction nexus: binding partners for connexins.

Gap junctions are the morphological correlates of direct cell-cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein-protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions.     

Pairing SOX off: with partners in the regulation of embryonic development

The SOX family of high-mobility group (HMG) domain proteins has recently been recognized as a key player in the regulation of embryonic development and in the determination of the cell fate. In the case of certain SOX proteins, they regulate the target genes by being paired off with specific partner factors. This partnering might allow SOX proteins to act in a cell-specific manner, which is key to their role in cell differentiation. The focus of this article is the mechanism of action of SOX proteins, in particular, how SOX proteins specifically pair off with respective partner factors and, as a consequence, select distinct sets of genes as their regulatory targets.     

Major Cdk5-dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis

Amphiphysin 1 (amph 1) is an endocytic protein enriched in the nerve terminals that functions in the clathrin-mediated endocytosis. It acts as membrane curvature sensor, a linker of clathrin coat proteins, and an enhancer of dynamin Guanosine Triphosphatase (GTPase) activity. Amph 1 undergoes phosphorylation by cyclin-dependent kinase 5 (Cdk5), at five phosphorylation sites, serine 262, 272, 276, 285, and threonine 310, as determined by mass spectrometry (MS). We show here that Cdk5-dependent phosphorylation of amph 1 is enhanced in the presence of lipid membranes. Analysis by tandem liquid chromatograph MS revealed that the phosphorylation occurs at two phosphorylation sites. The phosphorylation was markedly decreased by mutation either Ser276 or Ser285 of amph 1 to alanine (S276A and S285A). Furthermore, mutation of both sites (S276, 285A) completely eliminated the phosphorylation. Functional studies indicated that binding of amph 1 to lipid membrane was attenuated by Cdk5-dependent phosphorylation of wild type amph 1, but not of the S276, 285A form. Interestingly, endocytosis was increased in rat pheochromocytoma cells expressing amph 1 S276, 285A in comparison with wild type. These results suggest that Ser276 and Ser285 are regulatory Cdk5 phosphorylation sites of amph 1 in the lipid-bound state. Phosphorylation at these sites alters binding of amph 1 to lipid membranes, and may be an important regulatory aspect in the regulation of synaptic vesicle endocytosis.     

In vivo binding partners of the Lens culinaris lectin

The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.