Download Affinity purification of diverse plant and human 14-3-3

14-3-3 Proteins in Cell Regulation
Affinity purification of diverse plant and human 14-3-3-binding partners
F. C. Milne, G. Moorhead, M. Pozuelo Rubio, B. Wong, A. Kulma, J. E. Harthill, D. Villadsen, V. Cotelle
and C. MacKintosh'
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD I 5EH,
Scotland, U.K.
Abstract
the discovery that 14-3-3s inhibit phosphorylated
nitrate reductase [4,5]. This mechanism is triggered by a nutrient-sensing pathway that functions to inhibit nitrate assimilation in the dark, or
whenever photosynthesis is blocked. There are
at least two physiological reasons to co-ordinate
nitrate reductase activity with photosynthesis.
First, rates of nitrate assimilation must not exceed
the supplies of reductant or carbohydrate that are
required to combine with the reduced nitrogen to
form amino acids. Both the reductant and carbohydrate are produced by photosynthesis. Secondly, the product of nitrate reductase, nitrite, is very
toxic. Photosynthesis is required to produce the
reductant to convert nitrite into ammonia, which
means that there is a danger of nitrite poisoning in
the dark. Poisoning is prevented, however, by
rapid phosphorylation of nitrate reductase and
its inhibition by 14-3-3 proteins. In these ways,
14-3-3s can be considered to minimize the major
costs and potential hazards associated with nitrate
assimilation [6,7].
More recently, further plant proteins that
bind to 14-3-3s in a phosphorylation-dependent
manner have been identified, and it is striking that
many of these targets are involved in primary
sugar, amino acid and energy metabolism, including glutamine synthetases, an isoform of
trehalose-phosphate synthase, sucrose-phosphate
synthase and A T P synthase /3 [8-111. These
findings indicate that 14-3-3s are likely to play key
roles in determining the balance of carbon and
nitrogen assimilation, storage and distribution in
higher plants. T h e biochemical studies needed to
identify the relevant regulatory phosphorylation
and 14-3-3-binding sites on these proteins are
underway, preliminary to investigating the global
physiological functions of 14-3-3s in plant metabolism.
Many proteins that bind to a 14-3-3 column in
competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets
include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory
roles for 14-3-3s in partitioning carbon among the
pathways of sugar, amino acid, nucleotide and
protein biosynthesis in plants. Our results also
suggest that the current perception that 14-3-3s
bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen
because of the intensity of research on mammalian
signalling and for technical reasons.
14-3-3s in regulation of light-coupled
metabolism in plants
14-3-3s are a highly conserved family of proteins
that generally bind to phosphopeptide motifs in
diverse target proteins, and play central regulatory
roles in all eukaryotic cells. Recently, 14-3-3
proteins have emerged as components of the
signalling mechanisms that regulate light-coupled
metabolic fluxes in leaves.
Green leaves have the remarkable ability to
use A T P and reductants generated in the light
to fix CO, into sugars, and assimilate nitrate and
carbohydrate into amino acids. These lightcoupled biosynthetic processes are highly responsive to the environment and physiological status of
the plant [1,2]. Detailed measurements of enzyme
activities and metabolites have revealed complicated and shifting regulatory relationships among
the biosynthetic pathways. Subsets of metabolites
and enzymes activities display distinctive diurnal
variations, whose exact patterns are determined by
signals from phosphorylated sugars, p H sensors,
malate, nitrate and hormones [3].
T h e first identified role for 14-3-3s in the
regulation of light-coupled metabolism came with
14-3-3 affinity chromatography of
plant proteins
One procedure that proved very useful for identifying novel plant 14-3-3-binding proteins is
14-3-3 affinity chromatography, followed by
matrix-assisted laser-desorption ionization-time-
Key words 14-3-3 proteins, nitrate reductase, vesicle trafficking
'To whom correspondence should be addressed (e-mail
c rnackintosh(u1dundee ac uk)
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Biochemical Society Transactions (2002) Volume 30, part 4
differences in the types of 14-3-3-binding partners
found in the Plant and Animal Kingdoms seemed
odd, given the extremely high conservation of
14-3-3 sequences and the common mechanism
of interaction of 14-3-3s with phosphopeptide
motifs in target proteins.
Are 14-3-3s really specialized in signalling in
mammalian cells, or have perceptions been biased
by the intensity of research on mammalian signalling and for technical reasons? One potential
bias is that there are only a few reports of 14-3-3
proteins being used as bait in yeast two-hybrid
screens [15], although 14-3-3s are often picked up
with other proteins as bait (e.g. [16-201 and many
others). This may be because 14-3-3 proteins exist
as dimers and binding of 14-3-3 bait to endogenous or recombinant 14-3-3s can occur to the
exclusion of other interactions [21-231. Deleting
the dimerization site in the 14-3-3 bait may be
useful. Certainly, truncated monomeric forms of
14-3-3s can bind Raf-1 [24], although results from
yeast two-hybrid screens with monomeric 14-3-3s
as baits have not been reported widely. Interaction
cloning from bacterial expression libraries, which
of-flight (MALDI-TOF) tryptic mass fingerprinting of purified proteins (Figure 1) [8]. Generally, protein-protein chromatography is not
widely used because non-specific binding is a common problem. An important step in the 14-3-3
affinity chromatography procedure, though, is
the specific elution of bound proteins by competition with a synthetic phosphopeptide carrying
the canonical 14-3-3-binding motif, ARAApSAR
(where pS is phosphoserine). In this way, the pool
of purified proteins is highly enriched in proteins
that bind directly to the phosphopeptide-binding
site on 14-3-3s.
14-3-3 affinity purification of human
proteins of signalling, metabolism,
vesicle trafficking and chromatin
function
One reason that we were intrigued to find 14-3-3s
as regulators of enzymes in primary plant metabolism is that the majority of the known 14-3-3binding proteins in mammalian cells are enzymes
of oncogenic signalling pathways and cell-cycle
regulators (reviewed in [12-141). T h e apparent
Figure I
14-3-3 affinity chromatography
Cell and tissue extracts are prepared in buffers containing protein phosphatase inhibitorst o maintain the phosphorylation and 14-3-3-binding
capability of target proteins. The proportion of any particular target protein that binds t o the column depends on the proportion that is
phosphorylated under the conditions of cell extraction, and competition for the target protein between endogenous 14-3-3 proteins in the
extract and immobilized 14-3-3 proteins on the column. After extensive washing in salt and with control peptides, binding proteins are eluted
from the column with a synthetic 14-3-3-binding phosphopeptide. The elution pool contains proteins that bind directly and indirectly to
14-3-3s.Those proteins that bind directly to 14-3-3s can be identified by binding to 14-3-3s in overlay assays.
-
Plant/hurnan cell extract
14-3-3
column
‘=I
C
A -R-A- A- pS- A-P- A
Washes
NaCl 0.5M
Control peptides
Analys is
SOS-PAGE
14-3-3 overlays
Binding +/- dephosphorylation
Mass spec fingerprinting + Western blotting
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14-3-3 Proteins in Cell Regulation
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is another common method for identifying protein-protein interactions, is not suitable if 14-3-3binding proteins have to be phosphorylated.
Therefore, to determine whether or not
14-3-3s have related functions in both plants and
mammals, we applied 14-3-3 affinity chromatography to purify phosphorylated proteins from
human HeLa cells and bovine brain. Of course, it
may be that this method has its own bias, in that
it is easier to identify those purified proteins that
are more abundant. Nevertheless, we have recently purified many mammalian proteins by this
method. As the purified proteins are being identified and characterized, it seems striking that all the
proteins purified to date fall into four categories :
signalling components, enzymes of primary
metabolism, proteins of vesicle and protein trafficking, and proteins that regulate chromatin
function. Interestingly, the 14-3-3-affinity purified proteins do include mammalian counterparts
of known plant 14-3-3-binding proteins, such
as the mitochondria1 A T P synthase p subunit
(B. Wong, M. Pozuelo Rubio and C. MacKintosh,
unpublished work).
In addition to the evolutionary aspects, these
findings have other implications. For example,
from the phenotypes of Dvosophila and yeast
mutants, 14-3-3s have been implicated in neuronal
differentiation, learning and memory [25,26], regeneration of damaged tissue [27] and vesicle
trafficking [28,29]. T h e rationale for these phenotypes has often been discussed in terms of 14-3-3
interactions with signalling pathways. Our findings suggest that 14-3-3s regulate not only upstream signalling events but participate more
directly in cellular events such as vesicle trafficking
and metabolism.
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