12 research outputs found
eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and is Modulated by Phosphorylation
Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation
eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation
Analysis of the key elements of FFAT-like motifs identifies new proteins that potentially bind VAP on the ER, including two AKAPs and FAPP2.
Two phenylalanines (FF) in an acidic tract (FFAT)-motifs were originally described as having seven elements: an acidic flanking region followed by 6 residues (EFFDA-E). Such motifs are found in several lipid transfer protein (LTP) families, and they interact with a protein on the cytosolic face of the ER called vesicle-associated membrane protein-associated protein (VAP). Mutation of which causes ER stress and motor neuron disease, making it important to determine which proteins bind VAP. Among other proteins that bind VAP, some contain FFAT-like motifs that are missing one or more of the seven elements. Defining how much variation is tolerated in FFAT-like motifs is a preliminary step prior to the identification of the full range of VAP interactors
CDKA interacting proteins
Progression through the cell cycle relies on activity of cyclin-dependent kinases (CDKs) and their regulatory subunits, cyclins. CDKA and CDKB are the main components of the G2/M transition point in Arabidopsis thaliana. I aim to gain a better understanding of how mitotic kinases, CDKA and CDKB. mediate control of translation initiation and other biological processes throughout the cell cycle. To address this question, I used different approaches to identify interacting partners of CDKA and CDKB (and other cell cycle regulators). CDKA directly interacts with the complex that binds the 5' mRNA cap. In proliferating cells, CDKA associates with translation initiation factor eIF4A as demonstrated by reciprocal immonoprecipitation (Hutchins et al., 2004). To understand this interaction in greater depth, I used a pair-wise yeast two hybrid to test interactions between cell cycle regulators and translation initiation factors. I confirmed the association of translation initiation factors that is in agreement with the mechanism of cap complex assembly in animals and yeast and identified eIF4A and eIF4E as a putative substrates of both CDKA and CDKB2;1.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Targeting strength ratios (TSRs) and number of suboptimal residues for all the FFAT-motifs studied.
<p>The core FFAT-like motifs and immediate neighbours (6 amino-terminal and 2 carboxy-terminal) are shown for all sequences expressed in this study. Residues that might contribute to local charge (D, E, S, T, K or R) are in capitals, all others in lower case. Residues in bold indicate substitutions tested in this study. The number of other residues in the flanks of expressed constructs is also given. Full sequences and their precise origins are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone.0030455.s008" target="_blank">Table S5</a>.</p><p>“local ±” is the sum of charges in eight residues flanking the motif (six before and two after): K/R = +1 and D/E = –1.</p><p>“H” indicates that the region is predicted to be helical, <b>in bold</b> if known to be helical in structural studies (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone.0030455.s005" target="_blank">Table S2</a>).</p><p>“TSR” is the “Targeting Strength Ratio” measured from nuclear profiles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#s4" target="_blank">Materials & Methods</a>), indicating the strength of NE targeting.</p><p>“# sub-opt” is the number of sub-optimal elements in each motif, determined according to the method set out in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone.0030455.s005" target="_blank">Table S2</a>. Lower scores indicate a more optimal motif. Where the motif tested was multimerized the figures are in brackets, as they are not directly comparable.</p
The FFAT-like motif of Rab3GAP1 targets VAP.
<p>(A) 26 amino acids including the FFAT-like motif <sup>1</sup>EFFEC-S<sup>7</sup> from Rab3GAP1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-t001" target="_blank">Table 1</a>) were tagged with GFP and expressed in TLY251, which was grown in galactose to induce maximal yeast VAP (Scs2p) expression. Confocal sections through the plane of the nuclei showed the GFP construct interact with Scs2p, as fluorescence was found on the nuclear envelope (NE), in patches in the cell cortex, and in occasional strands in the cytoplasm, a pattern highly characteristic of the ER in yeast. Arrows indicate cells likely where the nucleus has been optically sectioned off the mid-line, so increasing central fluorescence and underestimating targeting. (B) As A, except GFP was tagged with the Px domain of Bem1p. This construct did not target membranes. (C) As A, except the FFAT-like sequence was altered at position 4: <sup>1</sup>EFF<b><u>A</u></b>C-S<sup>7</sup>. ER targeting is barely detectable, but much weaker. (D/E/F) Fluorescence was measured across nuclear profiles from (A) (B) and (C) respectively, (examples shown in the insets, above). Line scans were re-plotted to normalise for different nuclear widths, and profiles from five nuclei are shown.</p
FAPP2 in mammals has a weak FFAT motif.
<p>(A) An alignment of ≤60 a.a. from the region at the amino-terminus of 7 divergent GLTP sequences and the related region near the middle of 7 divergent FAPP2 sequences (see diagram, top). Three motifs are shown where the core <sup>2</sup>FFD<sup>4</sup> is partly conserved. (B) Diagram of the aligned sequences from (A), where the FFAT-like motifs are shown as logos, and those individual motifs that meet minimal criteria are shown as boxes, where darker shading indicates closer resemblance to the optimal motif (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone.0030455.s005" target="_blank">Tables S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone.0030455.s006" target="_blank">S3</a>). The best FFAT-like motif lies at different positions in different species. The most amino-terminal motif in human FAPP2 is quite weak. (C) 32 amino acids from FAPP2 in the opossum <i>M. domestica</i> including the FFAT-like motif <sup>1</sup>TFFSA-N<sup>7</sup> were expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-g001" target="_blank">Figure 1A</a>, together with variants <sup>1</sup> TFF<b><u>D</u></b>A-N<sup> 7</sup> and <sup>1</sup><b><u>E</u></b>FF<b><u>D</u></b>A-N<sup>7</sup>. While the wild-type (WT) sequence did not target, the variants with D<sup>4</sup> targeted weakly. (D) The dimer of 37 amino acids including the FFAT-like motif from human FAPP2 with two substitutions <sup>1</sup><b><u>E</u></b>FF<b><u>D</u></b>T-N<sup>7</sup> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-t001" target="_blank">Table 1</a>) were expressed as in (C), together with a dimer carrying a third change <sup>1</sup><b><u>E</u></b>FF<b><u>DA</u></b>-N<sup>7</sup>, and a tetramer of <sup>1</sup>TFF<b><u>D</u></b>T-N<sup>7</sup>. While the dimer with E<sup>1</sup>/D<sup>4</sup>/T<sup>5</sup> failed to target, the E<sup>1</sup>/D<sup>4</sup>/A<sup>5</sup> version targeted moderately, and the tetramer with D<sup>4</sup> targeted weakly.</p
FFAT-like motifs in two AKAPs.
<p>(A) 29 amino acids from AKAP220 including the FFAT-like motif <sup>1</sup>EFFDS-D<sup>7</sup> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-t001" target="_blank">Table 1</a>) were expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-g001" target="_blank">Figure 1A</a>, together with variants at position 5: <sup>1</sup>EFFD<b><u>E</u></b>-D<sup>7</sup> and <sup>1</sup>EFFD<b><u>A</u></b>-D<sup>7</sup>. The wild-type (WT) sequence showed moderate targeting, which was lost with E<sup>5</sup> and enhanced by A<sup>5</sup>. (B) 32 amino acids from AKAP110 including the FFAT-like motif <sup>1</sup>DFLTA-E<sup>7</sup> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-t001" target="_blank">Table 1</a>) were expressed as (A), together with variants at position 4: <sup>1</sup> DFL<b><u>D</u></b>A-E<sup> 7</sup> and <sup>1</sup> DFL<b><u>A</u></b>A-E<sup> 7</sup>. The wild-type (WT) sequence showed weak targeting, which was enhanced by D<sup>4</sup> and lost with A<sup>4</sup>.</p
FFAT-like motifs in plant ORPs.
<p>(A–D) Aligned FFAT-like motifs from (A) the subfamily of StART proteins related to Edr2 in <i>Arabidopsis thaliana</i> (<i>At</i>); as shown in the diagram, this region is between the StART domains and DUF1336, which are domains of unknown function structurally related to galectins. (B) ORPs. FFAT-like motifs, which occur upstream of ORP domains. Top: in <i>At</i> and homologues in other plants: <i>Gm – Glycine max; Os – Oryza sativa; Zm – Zea mays; Vv – Vitis vinifera; Pi – Petunia integrifolia</i>; bottom: in other species, as listed. (C) homologues of rabphilin-11from human, fish (one each) and <i>At</i> (x7). These motifs are at the extreme amino-termini. (D) Opi1p in the fungus <i>Ashbya gossypii</i>. (E) 34 amino acids from Orp1c (<i>Gm</i>) including the FFAT-like motif <sup>1</sup>AFFDT-D<sup>7</sup> were expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030455#pone-0030455-g001" target="_blank">Figure 1A</a>, together with variants <sup>1</sup>A<b><u>AA</u></b>DT-D<sup>7</sup> and <sup>1</sup><b><u>K</u></b>FFDT-D<sup> 7</sup>. The wild-type (WT) sequence showed moderate targeting, which was lost with <sup>2</sup>AA<sup>3</sup> and reduced with K<sup>1</sup>. (F) 32 amino acids from Orp2a (<i>At</i>) including the FFAT-like motif <sup>1</sup>SFHDT-E<sup>7</sup> were expressed as in (E) (left hand panel). We also expressed the dimer of this motif, and a dimer of a mutant: <sup>1</sup>S<b><u>AA</u></b>DT-E<sup>7</sup> (middle and right panels, images are at 2-fold lower magnification. The wild-type (WT) monomer failed to target, but moderate targeting was obtained upon dimerization, which was lost with <sup>2</sup>AA<sup>3</sup>.</p