Abstract
We have determined the absolute phosphate content of microtubule-associated proteins (MAPs) and established that phosphorylation inhibits the actin filament cross-linking activity of MAPs and both of the major MAP components, MAP-2 and tau. Similar results were obtained with actin from rabbit muscle, hog brain, and Acanthamoeba castellanii. We used the endogenous phosphatases and kinases in hog brain microtubule protein to modulate MAP phosphate level before isolating heat-stable MAPs. MAPs isolated directly from twice-cycled microtubule protein contain 7.1 +/- 0.1 (S.E.) mol of phosphate/300,000 g protein. After incubating microtubule protein without ATP, MAPs, had 4.9 +/- 0.6 phosphates. After incubating microtubule protein with 1 mM ATP and 5 microM cAMP in 2 mM EGTA, MAPs had 8.6 +/- 0.5 phosphates but there was also exchange of three more [32P]phosphates from gamma-labeled ATP for preexisting MAP phosphate. Incubation of microtubule protein with ATP and cAMP in 5 mM CaCl2 resulted in exchange but no net addition of phosphate to MAPs. We fractionated the MAP preparations by gel filtration and obtained MAP-2 with 4.3 to 7.5 and tau with 1.5 to 2.2 mol of phosphate/mol of protein depending on how we treated the microtubule protein prior to MAP isolation. The actin filament cross-linking activity of whole MAPs, MAP-2, and tau depended on the MAP-phosphate content. In all cases, phosphorylation of MAPs inhibited actin filament cross-linking activity. The concentration of high phosphate MAPs required to form a high viscosity solution with actin filaments was 2 to 4 times more than that of low phosphate. MAPs. During incubation of microtubule protein with [gamma-32P]ATP, only MAP peptides are labeled. Treatment of these MAPs with either acid or alkaline phosphatase removes phosphate mainly from MAP-2, with an increase in actin filament cross-linking activity. Thus, both MAP phosphorylation and the effect of phosphorylation on actin cross-linking activity of MAPs are reversible.
Highlights
MATERIALS AND METHODSPhosphoproteiPnhosphatasEe xperiments-Heat-stablMe APS (1.3to 2 mg/ml) in 100 mM KCl, 25 mM HEPESatp H7.5, plus 0.86
We report here experiments demonstrating that the extentFurthermore, the starting level of phosphorylation was not of phosphorylation of microtubule-associated proteins (MAPs)’ is one factor that can regulate
To produce MAPs with different phosphate contents,we followed sodium dodecyl sulfate[26].Samples were first dialyzed for 4h the procedures outlinedby Nishida et al [8].Microtubule proteinwas against distilled water, lyophilized, dissolved in the appropriate volincubated for 30 min a t 37 "C in buffer A with 2 mM EGTA or 5 mM ume of preheatedsample buffer containing 1.6% sodium dodecyl
Summary
PhosphoproteiPnhosphatasEe xperiments-Heat-stablMe APS (1.3to 2 mg/ml) in 100 mM KCl, 25 mM HEPESatp H7.5, plus 0.86. Microtubule protein was isolated from hog cerebral cortex by t,he of 452 mM KCl, 50 mM EGTA, 20 mMMgC12, 1 2 mM MES, 1 mM large scale isolation method described by Murphy [21] wherein 4 M HEPES, 0.4 mM ATP, pH 6.15, and 40 p1of 1.5 to 2.5 mg/ml actin glycerol is present only during the first polymerizationfrom brain solution in actin buffer. To produce MAPs with different phosphate contents,we followed sodium dodecyl sulfate[26].Samples were first dialyzed for 4h the procedures outlinedby Nishida et al [8].Microtubule proteinwas against distilled water, lyophilized, dissolved in the appropriate volincubated for 30 min a t 37 "C in buffer A with 2 mM EGTA or 5 mM ume of preheatedsample buffer containing 1.6% sodium dodecyl. Pg/ml pepstatin A, 2 pg/ml leupep- areas were quantitated by cuttingand weighing or by peak area tin, 2 pg/ml trypsin inhibitor, 2.5 mM dithiothreitol were added t o integration with a Hewlett-Packard digitizer
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