scholarly journals A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2)

1981 ◽  
Vol 90 (3) ◽  
pp. 568-576 ◽  
Author(s):  
RB Vallee ◽  
MJ DiBartolomeis ◽  
WE Theurkauf
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Gel Filtration ◽  
Microtubule Assembly ◽  
Protein Kinase Activity ◽  
Significant Activity ◽  
Gel Filtration Chromatography ◽  
Dependent Protein Kinase ◽  
Dependent Protein

In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consists of two structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures. MAP 2 is known to be specifically phosphorylated as the result of a protein kinase activity that is present in microtubule preparations. We have now found that the activity copurifies with the projection portion of MAP 2 itself. Kinase activity coeluted with MAP 2 when microtubule protein was subjected to either gel- filtration chromatography on bio-gel A-15m or ion-exchange chromatography on DEAE- Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface. The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on bio-gel A-15m. Three protein species (M(r) = 39,000, 55,000, and 70,000) cofractionated with MAP 2, and two of these (M(r) = 39,000 and 55,000) may represent the subunits of an associated cyclic AMP- dependent protein kinase. The projection-associated activity was stimulated 10-fold by cyclic AMP and was inhibited more than 95 percent by the cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle. It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAPs, or the assembly-promoting domain of MAP 2, and was estimated to account for 7-22 percent of the total brain cytosolic protein kinase activity. The location of the kinase on the projection is consistent with a role in regulating the function of the projection, though other roles for the enzyme are also possible.

scholarly journals Ovarian adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s). Regulation by choriogonadotropin and lutropin in rat ovarian cells

1976 ◽  
Vol 158 (2) ◽  
pp. 175-182 ◽  
Author(s):  
M R Clark ◽  
S Azhar ◽  
K M J Menon
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Gel Filtration ◽  
Supernatant Fraction ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Ovarian Cells ◽  
Dependent Protein

Choriogonadotropin and lutropin have been found to activate cyclic AMP-dependent protein kinase in ovarian cells isolated by collagenase dispersion from immature rats. The stimulatory effect of gonadotropins was dependent on both hormone concentration and incubation time. Choriogonadotropin at 1 mug/ml fully stimulated the protein kinase activity within 5 min of incubation, and this effect was specific for choriogonadotropin and lutropin-like activity. In addition, protein kinase activity has been characterized with respect to salt sensitivity, cyclic AMP binding, and its responsiveness to gonadotropins and other peptide hormones. Ovarian protein kinase was susceptible to high salt concentrations. The addition of 0.3-1.0 M-NaCl in incubation medium increased the activity ratio with a concomitant decrease in cycle AMP-dependence. The salt effect on protein kinase was observed both from hormone-treated and untreated cells. The hormone-stimulated and unstimulated protein kinase activity was completely stable in the absence of NaCl. No change in the activity ratio was observed when cellular extracts were assayed for protein kinase activity either immediately or after 2 h in the absence of added salt. Gel filtration in the absence of NaCl of cellular extracts prepared from choriogonadotropin-treated and untreated cells showned only a single peak of protein kinase activity that was sensitive to exogenously added cyclic AMP. By contrast, when 0.5 M-NaCl was included in the column buffer, the chromatography of untreated extract showed two peaks of protein kinase activity. The first peak was sensitive to added cyclic AMP, whereas the second peak was insensitive to it. Under identical experimental conditions, protein kinase from gonadotropin-treated cells showed, on gel filtration, only one peak of activity that was totally insensitive to added cyclic AMP. DEAE-cellulose column chromatography of a 20000 g supernatant fraction resulted in a peak of kinase activity that eluted in approx. 0.15 M-NaCl, similar to the similar to the elution of type II protein kinases as described by Corbin et al. (1975) (J. Biol. Chem. 250, 218-225). Choriogonadotropin stimulation produced a decrease in the capacity of protein kinase to bind exogenous cyclic [3H]AMP, with a concomitant increase in the kinase activity ratio. These results are consistent with the notion that cyclic AMP, GENERATED IN SITU Under hormonal stimulation, binds tot he regulatory subunit of protein kinase with subsequent dissociation of the active catalytic subunit from the holoenzyme.

scholarly journals The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

1973 ◽  
Vol 136 (4) ◽  
pp. 993-998 ◽  
Author(s):  
Malcolm C. Richardson ◽  
Dennis Schulster
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Adrenocorticotrophic Hormone ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Protein Kinase Activation ◽  
Dependent Protein

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of32P transferred from [γ-32P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1×10-2i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose–response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1×10-3i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.

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