Download Phosphorylation of apoproteins in VLDL, and LDL by protein

Biochemical SocietyTransactions ( 1 993) 21
Phosphorplation of apoproteins in VIADLand 1J)L by protein
kinases in vitro.
CLAIRE E HARRISON and MICHAEL R. MUNDAY
Dept of Pharmaceutical Chemistry, School of Pharmacy, University
of London, 29-39 Brunswick Square, London WClN IAX, UK.
Elevated plasma levels of cholesterol-rich low density
lipoprotein (LDL) are a major risk factor in the development of
atherosclerosis and coronary heart disease 111. LDL is formed from
intermediate density lipoprotein (IDL) derived from the peripheral
catabolism of very low density lipoprotein (VLDL) originally
assembled and secreted by liver 111. VLDL consists of a
triacylglycerol and cholesterol-ester core which is surrounded by a
monolayer of phospholipid interspersed with cholesterol. Embedded
in this monolayer is apoprotein E, a 34 KDa protein whose function
is to bind to LDL-receptorsand increase the plasma clearance of the
IDL 121, and apoprotein BlOO a 514 KDa protein that provides a
structuraJ framework for the partxle with alternating hydrophilic and
hydrophobic domains that interface with the aqueous medium and
the lipid core of the parr.de, respectively [3). Apo BlOO also forms
the receptor recognition site in LDL particles since these have lost
apoE during their formation from IDL [3].
Functional apo BlOO is essential for the hepatic
assembly and secretion of VLDL which is under hormonal control,
notably that of insulin and glucagon 141. There is evidence that
inhibibon of VLDL production is not always matched by a decrease
in apo B synthesis suggesting a possible post-translational
regulation of apo B function 14). It has been reported that in newlysecreted VLDL from rat liver or hepatocytes apo B is a
phosphoprotein 15, 61;that this phosphorylation is hormonally
regulated by insulin 16,qand is important for apo B function [7].
We have observed that CAMP-dependent protein kinase
(CAMP-PK),AMP-activated protein kinase (AMP-PK) and casein
kinase 2 stoichiometrically phosphorylate apo BlOO in vitro in
preparations of human LDL where it is the only apoprotein present
(M R Munday, D Carling, S Takhar and C E Harrison, unpublished
results). Incubation of human VLDL with [y-32P] ATP and the
catalytic subunit of bovine heart CAMP-PK in virro results in the
32P-labelling of a number of protein bands separated directly by
SDS PAGE (Figure la). One of these is apo BlOO in confirmation
of the results obtained with LDL. The major phosphorylated band
migmted with an apparent Mr of 35 KDa and minor phosphorylated
bands ran at 19 and 17 KDa. Other 32P-labelledproteins are a result
of phosphorylation of proteins within the protein kinase preparation
parhcularly the autophosphorylated C-subunit of CAMP-PK at 40
KDa (Figure la).
Figure la) Separation of VLDL apoproteins phosphorylated
by CAMP-PKin vitm on 8-15% SDS PAGE.
b) Cleveland V8 map on 15% SDS PAGE of apo B100
from I B L phosphorylated by CAMP-PKor AMP-PK in vitro.
a)
Rotein
Autoradiograph
blank VLDL blank VLDL
apoB
514 KDa
b) Autoradiograph
CAMP-PK AMP-PK
1295
Cleveland mapping of these protein bands using
Staphylococcus aurew V8 protease showed different patterns of
32P-labelled peptides for each suggesting that they were distinct
apoproteins and not proteolytic degradation products of apoB I00
(Figure 2). The 35 KDa band is suggestive of apoE whereas the
apparent 19 and 17 KDa bands fall midway between the quoted Mr
for apo A and apo C classes. Preliminary experiments suggest that
phosphorylations of VLDL by AMP-PK and casein kinase 2 result
in 32P-labelling of apo BlOO but not the other apoproteins (data not
shown).
Figure 2
Cleveland V8 map of 32P labelled apoprotein
bands from VLDL shown in Figure la.
apo BlOO
34 KDa (apo E)
17 KDa doublet
Cleveland V8 mapping of the apo BlOO band from the SDS
PAGE of LDL showed a different pattern of 32P-labelled peptides
for apo BlOO phosphorylated by CAMP-PKcompared with AMPPK (Figure lb). This indicates that these two kinases phosphorylate
different sites. This has also been suggested by reversed phase
HPLC separation of tryptic peptides from apo BlOO phosphorylated
by either kinase and will be confmed when these phosphorylation
sites are identified by amino acid sequencing.
The physiological significance of these phosphorylations is
unclear. Wr have observed that 32P-labelled apoproteins in VLDL or
LDL phosphorylated by CAMP-PKare rapidly dephosphorylatedon
subsequent incubation with plasma (known to be rich in phosphakx
activity). Therefore, if they are to be physiologically relevant, these
phosphorylationsare likely to be intracellular. Apo A, C and E are all
functionally important in the circulation, but apo B is most important
in intracellular VLDL assembly. Investigations of the topography of
VLDL assembly poses questions as to where apo BlOO may be
exposed to protein kinases 181. However, we have observed the
presence of up to 0.5 mol of alkali labile phosphate per mol of apo B
in freshly isolated human LDL. Phosphorylation of apo BlOO could
inhibit or augment VLDL assembly and secretion. The
phosphorylation of a number of proteins such as casein 191 and
vitellogenin 1101 is essential for their successful secretion.
Furthermore, hepatic CAMP-PK,AMP-PK and casein kinase 2 have
all been observed to be under the hormonal control of glucagon
andor insulin I11,12,131. Identification of apoprotein kinases, sites
of phosphorylation and their presence in the lipoproteins in vivo in
different physiological situations will reveal the importance of this
phenomenon to VLDL assembly, secretion and metabolism.
Claire Harrison is an SERC PhD student. We are grateful to the
British Heart Foundation for the financial support of this project.
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