Two Novel Src Homology 2 Domain Proteins Interact to Regulate Dictyostelium Gene Expression during Growth and Early Development

Christopher Sugden,Susan Ross,Gareth Bloomfield,Alasdair Ivens,Jason Skelton,Annette Mueller-Taubenberger,Jeffrey G Williams

doi:10.1074/jbc.m110.139733

Abstract

There are 13 Dictyostelium Src homology 2 (SH2) domain proteins, almost 10-fold fewer than in mammals, and only three are functionally unassigned. One of these, LrrB, contains a novel combination of protein interaction domains: an SH2 domain and a leucine-rich repeat domain. Growth and early development appear normal in the mutant, but expression profiling reveals that three genes active at these stages are greatly underexpressed: the ttdA metallohydrolase, the abcG10 small molecule transporter, and the cinB esterase. In contrast, the multigene family encoding the lectin discoidin 1 is overexpressed in the disruptant strain. LrrB binds to 14-3-3 protein, and the level of binding is highest during growth and decreases during early development. Comparative tandem affinity purification tagging shows that LrrB also interacts, via its SH2 domain and in a tyrosine phosphorylation-dependent manner, with two novel proteins: CldA and CldB. Both of these proteins contain a Clu domain, a >200-amino acid sequence present within highly conserved eukaryotic proteins required for correct mitochondrial dispersal. A functional interaction of LrrB with CldA is supported by the fact that a cldA disruptant mutant also underexpresses ttdA, abcG10, and cinB. Significantly, CldA is itself one of the three functionally unassigned SH2 domain proteins. Thus, just as in metazoa, but on a vastly reduced numerical scale, an interacting network of SH2 domain proteins regulates specific Dictyostelium gene expression.

Highlights

Interactions of SH22 domains with their phosphotyrosinecontaining binding sites are integral to many metazoan signal transduction pathways [1]
Because the ancestor of Dictyostelium diverged from the lineage leading to animals at some time after the divergence of ancestral plants [6], this implies a massive expansion in Src homology 2 (SH2) domain-based signaling during the evolution of the metazoa
To assess the importance of the SH2 domain in LrrB, lrrBϪ cells were transformed with tandem affinity purification (TAP)-tagged expression constructs containing either the unmutated lrrB gene or a mutant form, LrrB-CTAPmutR, in which arginine 198 of LrrB is substituted by alanine

Summary

EXPERIMENTAL PROCEDURES

Cell Culture, Transformation, Development, and Gene Disruption—Dictyostelium discoideum strain Ax2 was grown axenically and transformed as described [13, 14]. Cells were resuspended in 1 ml of TAP-Buffer A (10 mM Tris-Cl (pH 7.5 at 22 °C), 150 mM NaCl, 1% Nonidet P-40, 50 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 ␮g/ml pepstatin A, 1 mM benzamidine, 0.2 ␮M TLCK, and Complete protease inhibitor mixture (Roche Applied Science)) and incubated on ice for 3 min. The 14-3-3 pull- from vegetative cultures, washed in KK2, and resuspended down was normalized by quantitating LrrB-CTAP in the cor- and lysed in Lysis Buffer (50 mM Tris-Cl (pH 8.0 at 22 °C), 150 responding crude lysate by Western analysis with just the IRDyeTM 800 goat anti-rabbit IgG (H&L) (Rockland Immunochemicals), which detects the protein A portion of the TAP tag, and by quantitation of the LrrB-CTAP band. The lrrB primers were as follows: LrrB05 (5Ј-CTATGAAGAGAAAGTGCGAGTTATTTGG) (forward) and LrrB08 (5Ј-TCTCTTAAATCTTGCTCGATTGATG) (reverse); Ig7, 5Ј-TTACATTTATTAGACCCGAAACCAAGCG (forward) and Ig7rev (5Ј-TTCCCTTTAGACCTATGGACCTTAGCG) (reverse)

Gene Expression in the lrrB Disruptant Strain Is Aberrant during Growth

Gene name

LrrB Interacts with Two Very Large Related Proteins

DISCUSSION