Morderer D. Identification of Ca2+-dependent phosphorylation of scaffold protein ITSN1 and its interaction with cytoskeletal protein STOP in neurons

ITSN1 is a scaffold protein involved in formation of multimolecular complexes required for clathrin-mediated endocytosis, signal propagation through several intracellular signaling cascades and nucleation of actin filaments. Misregulation of ITSN1 expression is linked to several neurodevelopmental and neurodegenerative diseases such as Down syndrome, Alzheimer's disease and Huntington disease. Increasing evidence indicates that ITSN1 can be a factor implicated in development of these disorders. In support of this, ITSN1 was found to be involved in important neuronal functions such as dendritic spine development and synaptic vesicle recycling. The aim of this work was to identify new interacting partners of ITSN1 in neurons, evaluate its effect on the formation of dendritic tree by cultivated hippocampal neurons, and to test possible ITSN1 phosphorylation by Ca2+-dependent enzymes. In order to find new ITSN1-interacting proteins in neurons in vitro binding assay of ITSN1 SH3 domains with mouse brain lysate was performed. For SH3A domain we identified a 120 kDa band that was subjected to MALDI-TOF mass spectrometry analysis. This band was identified as STOP (stable tubule-only peptide) - microtubule-associated protein that mediates their stabilization. Interaction between ITSN1 and STOP was confirmed by in vitro binding assay of ITSN1 SH3 domains with mouse brain lysate and subsequent Western blot analysis. Furthermore, complexes of ITSN1 and STOP were precipitated from mouse brain lysate with anti-STOP and anti-ITSN1 antibodies, indicating their interaction in vivo. Using laser scanning confocal microscopy we showed that ITSN1 and STOP partially colocalize in rat primary hippocampal neurons. These findings indicate formation of complexes between ITSN1 and STOP in neurons. Neuronal cytoskeleton plays an important role in formation and growth of neurites and establishment of synaptic contacts. Recently STOP was shown to be important for dendritic tree formation. Since ITSN1 interacts with STOP and participates in actin regulation, we studied its role in dendritic development. We show that downregulation of ITSN by shRNA does not affect number of dendrites but results in decrease of total dendritic length. Also, reduction of length of dendrites without affecting their number and complexity was revealed by Sholl analysis. Thus, ITSN1 is involved in growth of dendrites. Ca2+ ions play an important role in neuron functions by participation both in propagation of electric impulses and in signal transduction within neuronal cell. Therefore, many neuronal proteins are in some way regulated by Ca2+ signaling system. One of possible ways to mediate the Ca2+ regulation is phosphorylation of proteins by Ca2+-dependent kinases. Using several bioinformatic tools we predicted the existence of 17 potential phosphorylation sites for Ca2+/calmodulin-dependent kinase 2 in ITSN1 molecule. Moreover, we demonstrate that particular fragments of ITSN1 can be phosphorylated in Ca2+/calmodulin-dependent manner. Using tandem mass spectrometry we identified 5 sites of Ca2+/calmodulin-dependent phosphorylation. Among them 2 were detected in CCR fragment, 1 - in EH-CCR interdomain linker, 1 - in SH3A-SH3B interdomain linker and 1 - in SH3B-SH3C interdomain linker.