Summary: | Protein kinase C (PKC) exists as a family of at least 10 (β1, β2 ,y,σ, ɛ,ɛ’,ξ, ƞ, L) phospholipid-dependent protein kinase activities. The PKC family may be
subdivided into two groups: group A (
α, β1, β2, y) which require free calcium ions
(Ca2+) in order to become fully active; and group B (σ, ɛ,ɛ’,ξ, ƞ, L) which may not.
Expression, and possibly subcellular localization, of each isoform varies with cell
type. The aim of the present study was to isolate, identify, and characterize the
Ca2-and phospholipid-dependent protein kinase activities from heart. In addition,
the presence of specific protein substrates in cardiac junctional and free
sarcoplasmic reticulum would be determined for the PKC subtype(s). Cardiac PKC
was isolated by chromatography on DEAE-Sephacel, phenyl-Sepharose and poly(L
lysine) Agarose columns. Estimates based upon enzyme recovery indicate the
presence of 10-20 mnollmin of protein kinase C activity per gram of bovine
ventricular myocardium. Hydroxylapatite column chromatography resolved the
preparation into two peaks of calcium- and phospholipid-dependent protein kinase
activity. By Western blot analysis, peaks 1
and 2 from the hydroxylapatite column
contained PKCβ (type II) and PKCα (type III), respectively. No cross-reactivity was
observed, indicating that separation was complete. PKCα, the major subtype
detected, was subsequently purified to apparent homogeneity by chromatography
on phosphatidylserine acrylamide. PKCβ activity could not be recovered following
phosphatidylserine affinity chromatography. Phosphoamino acid analysis showed
that PKCα autophosphorylated at a serine residue, whereas PKCβ
autophosphorylated at both serine and threonine residues. Among the various
phospholipids tested for activity, phosphatidylserine was the most effective. Both
subtypes were activated further by 1-stearoyl-2-arachidonylglycerol in the presence
of phosphatidylserine and calcium. Activation of both subtypes occured at free
calcium concentrations of less than 1
.µM. In addition to several similarities, these
two subtypes showed differences in activation and kinetic properties: PKCβ was
activated by cardiolipin, 1,2- and 1,3-dioleoylglycerol, and both cis- and trans
unsaturated fatty acids. PKCα was activated to a lesser degree by cardiolipin and
showed no response to 1,3-dioleoylglycerol. PKCα was activated to a greater extent
by 1,2-diacyiglycerols and by cis-unsaturated fatty acids. In the presence of
phosphatidylserine and 1-stearoyl-2-arachidonylglycerol, PKCβ exhibited
substantial activity in the presence of 1
mM ethyleneglycol-bis-(β-aminoethyl
ether)-N,N,N’,N’-tetraacetic acid (EGTA) without added calcium. Activation of
PKCβ and PKCα by unsaturated fatty acids was independent of phospholipid and
showed a lower apparent calcium affinity than observed for activation by
phosphatidylserine. These results show that cardiac protein kinase C isoforms β
and (
α
are functionally distinguishable and may play unique roles in the regulation
of cardiac function.
Protein phosphorylation is an important mechanism in the regulation of cardiac
function. Phosphoproteins are associated with the sarcolemma, contractile
apparatus, and sarcoplasmic reticulum (SR). The major phosphoprotein in the SR
is phospholamban: a 27 kDa homopentameric proteolipid, made up of 6,080-Da
subunits, which is believed to associate with and regulate the activity of the SR
calcium-transport ATPase. In the present study, canine cardiac SR vesicles were
separated into junctional (JSR) and free (FSR) fractions by phosphate-facilitated
calcium loading followed by centrifrigation into discontinuous sucrose density
gradients. Several potentially novel SR-associated polypeptides were observed in
this preparation. The substrate specificities of PKCα and PKCEβ, for
phosphorylation in junctional and free sarcoplasmic reticulum, were similar. In
addition, similar rates for the phosphorylation of phospholamban were observed for
these two isoforms. Two other proteins, enriched in the FSR, appeared to be
selectively phosphorylated by PKA. The identity of these two proteins and the
functional significance of this phosphorylation event are unknown. === Pharmaceutical Sciences, Faculty of === Graduate
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