SH3BGRL3 binds to myosin 1c in a calcium dependent manner and modulates migration in the MDA-MB-231 cell line

Filippo Di Pisa,Giovanni Renzone,Fabio Ghiotto,Silvia Bruno,Cinzia Bernardi,Maria Bono,Paolo Scartezzini,Franco Fais,Ermanno Ciccone,Elisa Pesenti,Andrea Scaloni,Andrea N Mazzarello,Michael P Lisanti

doi:10.1186/s12860-021-00379-1

Abstract

BackgroundThe human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members.We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability.ResultsWe first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent.A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility.ConclusionThe results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity.

Highlights

The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues
SH3BGRL3 protein binds to myosin 1c but not ErbB2 in SKBR3 cells The ErbB2-positive breast cancer cell line SKBR3, a widely used model to study ErbB2 expression, was chosen to assess the co-localization of SH3BGRL3 and ErbB2 proteins by confocal microscopy
We investigated the SH3BGRL3-ErbB2 interaction by co-immunoprecipitation (Co-IP); we set up dedicated experiments using both FLAG-SH3BGRL3 and ErbB2 as bait

Summary

Introduction

The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. The human SH3BGRL3 gene (SH3 Domain Binding Glutamate Rich Protein Like 3) ( named TIP-B1) was cloned by two independent groups [1, 2]. It belongs to a poorly characterized gene family, comprising three other members: SH3BGR, selectively expressed in muscle tissue [3, 4], and SH3BGRL [5] and SH3BGRL2 [6], that display ubiquitous expression. An ortholog of SH3BGRL3 gene is expressed in zebrafish, where it is already expressed in the early stages of embryonic development [7] These observations indicate a high degree of evolutionary conservation of the gene and suggest an important biological role for the SH3BGRL3 protein. While SH3BGR, SH3BGRL and SH3BGRL2 proteins contain the PLPP QIF sequence, which corresponds to the SH3 consensus sequence PXXPQ(L/I)(Y/F), SH3BGRL3 displays only a PPQIV sequence [1, 6]

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