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The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified as a microtubule protein kinase and as a microtubule protein phosphatase activator. FA could phosphorylate microtubule-associated tau protein up to 4 moles of phosphates per mole of protein. However, more than 80% of the phosphates in 32P-tau phosphorylated by FA could be removed by ATP.Mg-dependent protein phosphatase and the tau phosphatase activity was FA-dependent. Functional study further revealed that as a tau kinase, FA could phosphorylate tau and thereby inhibits cross-linking copolymerization of tau with tubulin and actin filaments whereas as a tau phosphatase activating factor, FA could promote copolymerization of tau with tubulin and actin filaments. Taken together, the results provide evidence that a cyclic modulation of cytoskeleton assembly-disassembly can be controlled by FA, representing an efficient cyclic cascade mechanism for rapid structural and functional regulation of cytoskeletal system in the central nervous system.

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factor FA has further been identified as a cAMP and Ca(2+)-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factor FA could phosphorylate synapsin I with a low Km value of about 2 microM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factor FA specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factor FA could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factor FA as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) was identified to exist in bovine retina. Furthermore, rhodopsin, the visual light pigment associated with rod outer segments in retina, could be well phosphorylated by kinase FA to about 0.9 mol of phosphates per mol of protein. Moreover, more than 90% of the phosphates in [32P]-rhodopsin could be completely removed by ATP.Mg-dependent protein phosphatase and the rhodopsin phosphatase activity was strictly kinase FA-dependent. Taken together, the results provide initial evidence that a cyclic phosphorylation-dephosphorylation of rhodopsin can be controlled by the retina-associated protein kinase FA, representing an efficient cyclic cascade mechanism possibly involved in the rapid regulation of rhodopsin function in retina.

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified as a protein kinase. The results are unexpected since factor FA possesses two activities which are antagonistic. As a kinase, factor FA catalyzes protein phosphorylation, while as a phosphatase activator, it catalyzes protein dephosphorylation. In this report, we found that the two opposing activities of factor FA could be selectively modulated. For instance, heparin at concentrations of 0.1-0.3 mg/ml could stimulate FA to work preferentially as a kinase towards phosphorylation of proteins but simultaneously inhibit it to work as a phosphatase activator towards dephosphorylation of the same proteins. In a similar manner, alkaline pH could stimulate FA to work as a kinase but block it to work as a phosphatase activator. This is the first report providing initial evidence that the two opposing activities of factor FA can be selectively modulated in a reciprocal manner by various triggers, suggesting that a simultaneous coordinate control mechanism may well be involved in regulating the activities of factor FA in the cell.

The activating factor of ATP.Mg-dependent protein phosphatase (FA) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, FA could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a Km value of 0.4 microM, and tau proteins to 4 moles of phosphates per mole of proteins with a Km value of about 3 microM. When using microtubules as substrates, FA could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, the FA-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by FA. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that FA could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to FA. Taken together, the results provide initial evidence that the ATP.Mg-dependent protein phosphatase activating factor (FA) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32P-labeled myelin basic protein (MBP) and [32P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues.

Exposure of rat adipocytes to physiological concentrations of insulin resulted in a time- and concentration-dependent activation-translocation of kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) in plasma membranes and the subsequent activation of ATP.Mg-dependent protein phosphatase in the cytosol. The insulin-induced activation of membrane-associated kinase FA and cytosolic ATP.Mg-dependent protein phosphatase occurred very rapidly, reaching the maximal activity levels within 3 min. Moreover, the insulin effect is transient; the insulin-stimulated FA activity in membranes and ATP.Mg-dependent phosphatase activity in the cytosol returned to control levels within 30 min. It is concluded that insulin may induce the activation of kinase FA in membranes and thereby promotes the activation of ATP.Mg-dependent multifunctional protein phosphatase in the cytosol of rat adipocytes in order to mediate some of its intracellular effects through the dephosphorylation reactions. The release of factor FA from plasma membranes may represent one of the transmembrane signalling mechanisms for insulin actions.

The cytosolic fractions from epidermal growth factor (EGF)-treated A431 cells exhibit a marked increase in activities of ATP.Mg-dependent protein phosphatase and its activating factor (protein kinase FA) when compared to controls in the absence of EGF. By contrast, the Triton X-100-solubilized membrane fractions from the same EGF-treated cells exhibit a corresponding decrease in protein kinase FA activity. The EGF-dependent activation of protein kinase FA and ATP.Mg-dependent protein phosphatase occurred within physiological concentrations of EGF (ED50 = 5 x 10(-10) M). The changes of kinase and phosphatase activities which were measured concomitantly exhibit very similar characteristics as to EGF sensitivity and time dependence. The EGF-induced kinase and phosphatase activation occurred very rapidly, reaching the maximal activity levels within 3 min. Moreover, the EGF effect is transient; both EGF-stimulated phosphatase and kinase activities returned to control levels within 30 min. Taken together, the results suggest that EGF may induce the activation of kinase FA in the membrane and thereby promotes the activation of ATP.Mg-dependent phosphatase in the cytosol. Exposure of A431 cells to exogenous phospholipase C also resulted in the activation of endogenous kinase FA and ATP.Mg-dependent phosphatase in a similar pattern produced by EGF. This further suggests that phospholipase C can mimic EGF to mediate the activation of kinase FA and ATP.Mg-dependent phosphatase in A431 cells. By its dual role as a multisubstrate protein kinase and as an activating factor of multisubstrate protein phosphatase, protein kinase FA may represent a transmembrane signal of EGF.

An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.

A protein (FA) has been isolated from rabbit muscle which has two functions: one is the activation of the ATP x Mg-dependent phosphatase (see previous paper) (1) and the second is the phosphorylation and concomitant inactivation of glycogen synthase, independent from cyclic AMP or Ca ions. The two activities co-purify throughout the purification scheme, and reside in the single protein band that the purified preparation shows in discontinuous acrylamide gel electrophoresis. Heat inactivation experiments with the purified protein showed a parallel decrease of both activities with time. GTP could efficiently replace the ATP in both reactions. Sodium dodecyl sulfate-gel electrophoresis also shows a single protein-stained band corresponding to a Mr = approximately 50,000 and sucrose density gradient centrifugation gave a value of 45,000. The enzyme incorporates only 1 mol of phosphate/mol of synthase monomer (85,000 daltons) and brings the activity ratio (+/- glucose-6-P) down to less than 0.05. Kinetic studies suggest that FA exerts its two activities in quite different ways: the activation of the ATP x Mg-dependent phosphatase is bought about by a protein-protein interaction (FA x FC complex formation) with ATP x Mg as a necessary cofactor, whereas for the inactivation of synthase, FA is a cyclic AMP- and Ca-independent kinase.

The mechanism of activation of the Mg(II) X ATP-dependent phosphatase by the kinase FA has been investigated. The inactive protein phosphatase can be represented as FC X M where FC is the inactive catalytic component and M is the heat-stable modulator protein (also known as inhibitor-2). Phosphorylation of the modulator protein is demonstrated during activation of FC X M. In addition, continuous ATP hydrolysis during the activation is observed. This suggests that a cyclic phosphorylation-dephosphorylation reaction is continuously occurring during the activation. It is proposed that phosphorylation of the modulator protein causes an isomerization in FC to generate an active phosphatase. The activated phosphatase is capable of dephosphorylating the phosphorylated modulator. Upon dephosphorylation of modulator, the active phosphatase returns to its inactive form via a slow isomerization.