Characterization of Functional Domains of Human EB1 Family Proteins

Li-Kuo Su,Wen Bu

doi:10.1074/jbc.m306194200

Abstract

EB1 family proteins are evolutionarily conserved proteins that bind microtubule plus-ends and centrosomes and regulate the dynamics and organization of microtubules. Human EB1 family proteins, which include EB1, EBF3, and RP1, also associate with the tumor suppressor protein adenomatous polyposis coli (APC) and p150glued, a component of the dynactin complex. The structural basis for interaction between human EB1 family proteins and their associated proteins has not been defined in detail. EB1 family proteins have a calponin homology (CH) domain at their N terminus and an EB1-like C-terminal motif at their C terminus; the functional importance of these domains has not been determined. To better understand functions of human EB1 family proteins and to reveal functional similarities and differences among these proteins, we performed detailed characterizations of interactions between human EB1 family proteins and their associated proteins. We show that amino acids 1-133 of EB1 and EBF3 and the corresponding region of RP1, which contain a CH domain, are necessary and sufficient for binding microtubules, thus demonstrating for the first time that a CH domain contributes to binding microtubules. EB1 family proteins use overlapping but different regions that contain the EB1-like C-terminal motif to associate with APC and p150glued. Neither APC nor p150glued binding domain is necessary for EB1 or EBF3 to induce microtubule bundling, which requires amino acids 1-181 and 1-185 of EB1 and EBF3, respectively. We also determined that the EB1 family protein-binding regions are amino acids 2781-2820 and 18-111 of APC and p150glued, respectively.

Highlights

Microtubules are important for many cellular functions, such as intracellular transportation, maintenance of cell shape and cell polarity, and chromosome segregation
We show that amino acids 1–133 of EB1 and EBF3 and the corresponding region of RP1, which contain a calponin homology (CH) domain, are necessary and sufficient for binding microtubules, demonstrating for the first time that a CH domain contributes to binding microtubules
Human EB1 was initially identified as a protein that interacts with the C-terminal portion of the adenomatous polyposis coli (APC)1 tumor suppressor protein [5]

Summary

TABLE I Primers used for generating cDNA fragments by PCR

AGCAACAAGGGAAGGAGC AAGCTTATTGACCTTGTCTGGCAGCCACA AAGCTTATTGACCTTGTCTGGCAGCCACA AAGCTTAATCGAAAAACTTCTTG CTCGAGTTATCGCACCACACCAGG AAGCTTTTAATACTCTTCTTGCTCCTCC GCAAGCTTGGGCCCCCTTCATCAGG AAGCTTATTCATCTGTGGCATACAG GCGTCGACCTCTGCAATACAGGGTCG AAGCTTTTAATACTCTTCTTGCTCCTCC AAGCTTATTCATCTGTGGCATACAG AAGCTTTTAATACTCTTCTTGCTCCTCC AAGCTTATTCATCTGTGGCATACAG AAGCTTTTAATACTCTTCTTGCTCCTCC GCAAGCTTCTTGCTCCTCCTGTGG GCGTCGACCTCTGCAATACAGGGTCG AAGCTTCACTGGCCCTGCCGCGC AAGCTTCACTGGCCCTGCCGCGC ATCCTTTCCATCATAGTTTGC AAGCTTCCCTCAGTACTCGTCCTGG AAGCTTTCAGCCTGTGGGGGACGTCCTC CTCGAGTCACCGGAGAATGCAGGG AAGCTTATTCCTCTGTGGCATAGAG AAGCTTCATTGCCCTTGTCGTGC AAGCTTCATTGCCCTTGTCGTGC AAGCTTATTCTTCTGAAGCATACAG AAGCTTTTAGGTTCCACTGGATTCTGTG AAGCTTTTAGGTTCCACTGGATTCTGTG AAGCTTTTAGGTTCCACTGGATTCTGTG AAGCTTTTAGGTTCCACTGGATTCTGTG AAGCTTTTAGGTTCCACTGGATTCTGTG AAGCTTAAGTTTTGGAATCTCGCTTC AAGCTTAATTCACTGGAGTTGGGATC AAGCTTAAGTTTTGGAATCTCGCTTC AAGCTTAAGAATCAGGTGTCTCTGG AAGCTTAGATGCCATGCCCTTCATC a The APCd7 was generated by digesting the PCR product using BglII, which cut at codon 2763, and HindIII, which cut at the 3Ј primer. EB1 family proteins associate with microtubules and can induce microtubule bundling, they do not contain any previously characterized microtubule-binding domains. All three human EB1 family proteins express ubiquitously, but most studies so far have focused on EB1. To better understand functions of human EB1 family proteins and to reveal potential similar and different functions of these proteins, we performed detailed characterization of interactions between human EB1 family proteins and their associated proteins

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION