ABSTRACT
Actin polymerization plays a critical role in clathrin-mediated endocytosis in many cell types, but how polymerization is regulated is not known. Hip1R may negatively regulate actin assembly during endocytosis because its depletion increases actin assembly at endocytic sites. Here, we show that the C-terminal proline-rich domain of Hip1R binds to the SH3 domain of cortactin, a protein that binds to dynamin, actin filaments and the Arp2/3 complex. We demonstrate that Hip1R deleted for the cortactin-binding site loses its ability to rescue fully the formation of abnormal actin structures at endocytic sites induced by Hip1R siRNA. To determine when this complex might function during endocytosis, we performed live cell imaging. The maximum in vivo recruitment of Hip1R, clathrin and cortactin to endocytic sites was coincident, and all three proteins disappeared together upon formation of a clathrin-coated vesicle. Finally, we showed that Hip1R inhibits actin assembly by forming a complex with cortactin that blocks actin filament barbed end elongation.
Subject(s)
Actins/metabolism , Cortactin/metabolism , Endocytosis/physiology , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Cortactin/genetics , Cortactin/pharmacology , Dynamins/metabolism , Endocytosis/drug effects , HeLa Cells , Humans , Huntingtin Protein , Microfilament Proteins , Models, Biological , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Protein Binding , RNA Interference , Signal Transduction/drug effects , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/pharmacologyABSTRACT
Cyclin G-associated kinase (GAK), also known as auxilin 2, is a potential regulator of clathrin-mediated membrane trafficking. It possesses a kinase domain at its N-terminus that can phosphorylate the clathrin adaptors AP-1 and AP-2 in vitro. The GAK C-terminus can act as a cochaperaone in vitro for Hsc70, a heat-shock protein required for clathrin uncoating. Here we show that the specificity of GAK is very similar to that of adaptor-associated kinase 1, another mammalian adaptor kinase. We used siRNA to investigate GAK's in vivo function. We discovered that early stages of clathrin-mediated endocytosis (CME) were partially inhibited when GAK expression was knocked down. This defect was specifically caused by GAK knockdown because it could be rescued by expressing a rat GAK gene that could not be silenced by one of the siRNAs. To identify the GAK activity required during CME, we mutated the kinase domain and the J domain of the rat gene. Only GAK with a functional J domain could rescue the defect, suggesting that GAK is important for clathrin uncoating. Furthermore, we demonstrated that GAK plays a role in the clathrin-dependent trafficking from the trans Golgi network.
Subject(s)
Clathrin/physiology , Cyclins/physiology , Protein Serine-Threonine Kinases/physiology , Animals , Cathepsin D/metabolism , Cyclins/deficiency , Cyclins/genetics , HeLa Cells , Humans , Lysosomes/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , RNA Interference , Rats , Threonine/genetics , Threonine/metabolism , Transferrin/metabolism , trans-Golgi Network/metabolismABSTRACT
Actin filaments transiently associate with the endocytic machinery during clathrin-coated vesicle formation. Although several proteins that might mediate or regulate this association have been identified, in vivo demonstration of such an activity has not been achieved. Huntingtin interacting protein 1R (Hip1R) is a candidate cytoskeletal-endocytic linker or regulator because it binds to clathrin and actin. Here, Hip1R levels were lowered by RNA interference (RNAi). Surprisingly, rather than disrupting the transient association between endocytic and cytoskeletal proteins, clathrin-coated structures (CCSs) and their endocytic cargo became stably associated with dynamin, actin, the Arp2/3 complex, and its activator, cortactin. RNAi double-depletion experiments demonstrated that accumulation of the cortical actin-endocytic complexes depended on cortactin. Fluorescence recovery after photobleaching showed that dynamic actin filament assembly can occur at CCSs. Our results provide evidence that Hip1R helps to make the interaction between actin and the endocytic machinery functional and transient.
Subject(s)
Actins/chemistry , Actins/metabolism , DNA-Binding Proteins/genetics , Endocytosis , Actin Cytoskeleton/chemistry , Actin-Related Protein 2 , Actin-Related Protein 3 , Adaptor Proteins, Signal Transducing , Blotting, Western , Cell Nucleus/metabolism , Cortactin , Cytoskeletal Proteins/metabolism , DNA/chemistry , Dynamins/chemistry , Fluorescent Antibody Technique, Indirect , Gene Silencing , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/metabolism , Microfilament Proteins/chemistry , Microscopy, Fluorescence , Phenotype , Propidium/pharmacology , RNA/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Time Factors , Transferrin/chemistry , Transferrin/pharmacokinetics , Vesicular Transport ProteinsABSTRACT
Internalization of receptors, lipids, pathogens, and other cargo at the plasma membrane involves several different pathways and requires coordinated interactions between a variety of protein and lipid molecules. The actin cytoskeleton is an integral part of the cell cortex, and there is growing evidence that F-actin plays a direct role in these endocytic events. Genetic studies in yeast have firmly established a functional connection between actin and endocytosis. Identification of several proteins that may function at the interface between actin and the endocytic machinery has provided further evidence for this association in both yeast and mammalian cells. Several of these proteins are directly involved in regulating actin assembly and could thus harness forces produced during actin polymerization to facilitate specific steps in the endocytic process. Recent microscopy studies in mammalian cells provide powerful evidence that localized recruitment and polymerization of actin occurs at endocytic sites. In this review, we focus on progress made in elucidating the functions of the actin cytoskeleton in endocytosis.
