Journal of Neuroscience, Vol 6, 3618-3627, Copyright © 1986 by Society for Neuroscience
Phosphoprotein F1: purification and characterization of a brain kinase C substrate related to plasticity
SY Chan, K Murakami and A Routtenberg
To study the role of protein kinase C (PKC) and its substrates in neuronal
function, we have investigated the in vitro endogenous phosphorylation of
the neuronal phosphoprotein F1 after induction of synaptic plasticity by
long-term potentiation (LTP). The protein F1 phosphorylation was found to
increase 5 min (Routtenberg et al., 1985), 1 hr (Lovinger et al., 1986) and
3 d (Lovinger et al., 1985) after LTP. The characteristics of this protein
bear close similarities to a number of proteins characterized in various
neuronal systems, such as B50 (brain specific, synaptosome-enriched
protein), pp46 (a growth cone protein), and GAP 43 (nerve growth and
regeneration-associated protein). A positive identification of the purified
protein F1 with these proteins would link protein F1 to the developmental
growth of axons, nerve regeneration, and polyphosphoinositide metabolism,
as well as adult plasticity. We have therefore purified and partially
characterized native protein F1 so that a meaningful comparison among the
properties of these proteins can be made. Using synaptosomal plasma
membrane (P2') as starting material, subsequent purification involved pH
extraction, 40-80% ammonium sulfate precipitation, hydroxylapatite, and
phenyl-Sepharose column chromatography. This procedure achieved greater
than 800-fold purification and about 45% yield relative to P2'. Purified
protein F1 (Mr = 47,000, pI = 4.5) was found to be a hydrophilic molecule
and was phosphorylated by 1000-fold purified PKC in the presence of
phosphatidylserine (PS) and Ca2+. The Ka of PS activation is about 15
micrograms/ml (approximately 20 microM), and that of Ca2+ is about 25
microM. Diolein and DiC:8 (a synthetic diacylglycerol) lowered the
requirement of Ca2+ for maximal stimulation from 100 to 5 microM.
Ca2+-calmodulin kinases type I and II did not phosphorylate protein F1. The
phosphoamino acid analysis showed that 97% of the total incorporated
32P-phosphate was on the serine residue. Phosphopeptide mapping using
V8-protease generated 2 phospho-fragments having apparent Mr of 13,000 and
11,000. Calmodulin at 3.6 microM inhibited 95% of protein F1
phosphorylation by PKC. The availability of purified native protein F1
should facilitate investigation of the physiological role of this protein
in the nervous system and its functional regulation by PKC.
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