P104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell

Ki Young Choi,Seung Hwan Hong,Min Sup Lee,Myong Ho Jeong,Seung Jin Han,Young Jun Cho

doi:10.1155/2014/592450

Ki Young Choi, Seung Hwan Hong + Show 4 more

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https://doi.org/10.1155/2014/592450

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Abstract

The p104 protein inhibits cellular proliferation when overexpressed in NIH3T3 cells and has been shown to associate with p85α, Grb2, and PLCγ1. In order to isolate other proteins that interact with p104, yeast two-hybrid screening was performed. Rac1 was identified as a binding partner of p104 and the interaction between p104 and Rac1 was confirmed by immunoprecipitation. Using a glutathione S-transferase (GST) pull-down assay with various p104 fragments, the 814–848 amino acid residue at the carboxyl-terminal region of p104 was identified as the key component to interact with Rac1. The CrkII which is involved in the Rac1-mediated cellular response was also found to interact with p104 protein. NIH3T3 cells which overexpressed p104 showed a decrease of Rac1 activity. However, neither the proline-rich domain mutant, which is unable to interact with CrkII, nor the carboxy-terminal deletion mutant could attenuate Rac1 activity. During the differentiation of myoblasts, the amount of p104 protein as well as transcript level was increased. The overexpression of p104 enhanced myotube differentiation, whereas siRNA of p104 reversed this process. In this process, more Rac1 and CrkII were bound to increased p104. Based on these results, we conclude that p104 is involved in muscle cell differentiation by modulating the Rac1 activity.

Highlights

The p104, a cell proliferation regulator, was initially identified as a binding protein of phospholipase Cγ1 (PLCγ1)
Overexpression of p104 leads to increased levels of p27kip1, a cyclindependent kinase inhibitor, and it inhibits the activities of phosphoinositide 3-kinase (PI3K) that are required for cellular proliferation [1]
GTPases cycle between two conformational states; GTP(“active” state) and GDP-bound forms (“inactive” state), by hydrolyzing GTP to GDP. The transition between these two states is tightly regulated by three distinct families of proteins including guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and the guanine nucleotide dissociation inhibitors (GDIs)

Summary

Introduction

The p104, a cell proliferation regulator, was initially identified as a binding protein of phospholipase Cγ1 (PLCγ1) It can interact with the p85α subunit of phosphoinositide 3-kinase (PI3K) via its proline-rich (PXXP) motifs [1]. GTPases cycle between two conformational states; GTP(“active” state) and GDP-bound forms (“inactive” state), by hydrolyzing GTP to GDP The transition between these two states is tightly regulated by three distinct families of proteins including guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and the guanine nucleotide dissociation inhibitors (GDIs). The aminoterminal SH3 domain of the CrkII interacts with several proteins that contain a PXXPXK binding motif, including C3G (a nucleotide exchange factor for Rap1), DOCK180, as well as the Abl tyrosine kinase, tyrosine phosphatase, the p85 subunit of PI3K, and c-Jun N-terminal kinase (JNK) [37,38,39]. Rac was identified as a new interacting partner of p104 using a yeast two-hybrid screening system. p104 was shown to be involved in reducing the activity of Rac and implicated in myotube differentiation of C2C12 cells by regulating Rac activity

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