Philip L. Mobley

Protein kinase C in astrocytes: a determinant of cell morphology

Protein kinase C-like activity was found to be present in astrocytes prepared from rat neocortex and maintained in culture. Exposure to phorbol 12-myristate 13-acetate (PMA) caused a redistribution of this kinase from the cytosol to the membrane fraction of these cells. Also PMA was found to cause a profound change in astrocyte morphology; cells were converted from flat, polygonal, undifferentiated cells to process-bearing cells.

Phosphorylation of glial fibrillary acidic protein and vimentin by cytoskeletal-associated intermediate filament protein kinase activity in astrocytes

Abstract: These studies describe a cytoskeletal‐associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK‐C) and the cyclic AMP‐dependent protein kinase (PK‐A). The cytoskeletal‐associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mMMgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK‐C and PK‐A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium‐dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin‐dependent kinase activity acts mainly at serine residues. PK‐A and PK‐C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N‐and the C‐terminal domains of vimentin and the N‐terminal domain of GFAP. In contrast, both PK‐C and PK‐A are known to phosphorylate the N‐terminal domains of both proteins. The intermediate filament protein kinase activity appeared to be distinct from that of PK‐C and PK‐A and was able to phosphorylate unique sites of intermediate filament proteins. Also, it appeared that the intermediate filament protein kinase activity represents two kinases: a magnesium‐dependent protein kinase and a calcium/calmodulin‐dependent protein kinase.

Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Abstract: Both the protein kinase C (PK‐C) activator, phorbol 12‐myristate 13‐acetate (PMA), and the cyclic AMP‐dependent protein kinase (PK‐A) activator, 8‐bromo‐cyclic AMP (8‐BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8‐BR results in the morphological transformation of flat, polygonal‐shaped cells into stellate, process‐bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8‐BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8‐BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8‐BR treatment was restricted to serine residues in the N‐terminal domain of these proteins. Studies were also conducted to compare the two‐dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8‐BR with those produced when the proteins were phosphorylated with purified PK‐C or PK‐A. PK‐C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK‐C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells. The increased phosphorylation of vimentin observed in intact cells treated with 8‐BR and with PK‐A using purified vimentin as a substrate resulted in similar phosphopeptide maps. A common major phosphorylated peptide fragment was observed following phosphorylation of GFAP by PK‐A and in intact cells treated with 8‐BR; however, an additional phosphorylated fragment was observed with the purified kinase that was not observed in intact cells. These studies indicate that phosphorylation events mediated by PMA and 8‐BR converge to phosphorylate several of the same regions of GFAP and vimentin, and suggest that these effects are the direct result of the activation of PK‐C and PK‐A.

Protein phosphorylation in astrocytes mediated by protein kinase C: comparison with phosphorylation by cyclic AMP-dependent protein kinase.

Abstract: The protein kinase C activator, phorbol 12‐myris‐tate 13‐acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate‐shaped, process‐bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP‐dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP‐dependent mechanisms.

Astrocyte morphology altered by 1-(5-isoquinolinylsulfonyl) 2-methyl piperazine (H-7) and other protein kinase inhibitors.

Studies were conducted to determine if the protein kinase C inhibitor H-7 could block the effects of phorbol-12-myristate-13-acetate (PMA) on astrocyte morphology. Contrary to expectation, H-7 alone was found to induce morphological changes very similar to those elicited by PMA. This effect was shared by two other inhibitors of protein kinase C, H-8 and staurosporine, but not by the cyclic nucleotide-dependent protein kinase inhibitor HA-1004 or the calcium/calmodulin dependent protein kinase inhibitor W-7. Although the morphological effects observed with H-7 resemble those induced by PMA, H-7 did not promote the redistribution of protein kinase C to the membrane or induce the phosphorylation of endogenous proteins like PMA. In addition, the effects of H-7 were still observed in cells depleted of protein kinase C activity which were no longer responsive to treatment with PMA. Cytoskeletal elements appear to be involved in the effect of H-7 on cell shape since this effect is blocked by treatment with colchicine. Activators of the cyclic AMP-dependent protein kinase also alter astrocyte shape, however, while H-7 did cause a slight increase in cyclic AMP levels, it was unlikely that this action is responsible for its effect on morphology. One common action of both H-7 and PMA was to decrease the 32P content of several 20,000 Da proteins. While the mechanism by which H-7 exerts its influence on astrocyte morphology remains to be clarified, be it by the inhibition of protein kinase C or some other mechanism, the results suggest that caution must be used when interpreting the effects of activators and inhibitors of this kinase.

Norepinephrine-mediated protein phosphorylation in astrocytes.

Studies were conducted to determine if norepinephrine activates both protein kinase C and the cyclic AMP-dependent protein kinase in cultured rat astrocytes using phosphoproteins as markers. Norepinephrine was found to decrease 32P incorporation into an acidic 80,000 M(R) protein. A similar response was observed with isoproterenol and cyclic AMP analogs. In contrast, phorbol myristate acetate (PMA) increased 32P incorporation into this protein. Further studies looked at phosphorylation sites on glial fibrillary acidic protein and vimentin using two-dimensional tryptic phosphopeptide maps. The pattern of phosphorylation of these two proteins by norepinephrine resembles that of 8-bromo cyclic AMP and isoproterenol, and not that of PMA. Additionally, the effect of norepinephrine on the phosphorylation of GFAP and vimentin was blocked by alprenolol. One difference noted between norepinephrine and isoproterenol was the phosphorylation of an 18,000 M(R) protein. Norepinephrine increased, and isoproterenol decreased, 32P incorporation into this protein; however, the mechanism which mediates the norepinephrine effect remains to be determined. Overall, these studies indicate that the most prominent phosphorylation events mediated by norepinephrine are the consequence of the activation of cyclic AMP-dependent protein kinase.

The use of microwave tissue fixation to demonstrate the in vivo phosphorylation of an acidic 80,000 molecular weight protein in the rat neocortex following treatment with soman

Studies were conducted to determine if soman, a cholinesterase inhibitor, could activate the protein kinase C system in the rat neocortex. Using microwave radiation for rapid tissue fixation, it was demonstrated that treatment with soman increased 32P incorporation into an acidic 80,000 molecular weight, heat-stable protein in vivo. Based on relative molecular weight and isoelectric point this protein appears to be identical to a protein identified as a substrate for protein kinase C. Additionally, a protein of the same molecular weight and isoelectric point could be phosphorylated in tissue slices prepared from the neocortex by cholinergic dependent mechanisms. Also, treatment with soman decreased protein kinase C in the soluble fraction of this brain region; however, no corresponding increase was observed in the particulate fraction. These results suggest that soman can activate protein kinase C in vivo, and demonstrate the utility of using microwave tissue fixation to study protein phosphorylation events in vivo.