Effects of Phosphorylation

It is well known that phosphorylation activates the MgATPase activity of smooth muscle myosin (see 

Chapter 1, this volume). Phosphorylation is also essential for movement of actin filaments in both the Nitella and the sliding actin in vitro motility assays (Umemoto and Sellers, 1990 Warshaw etai, 1990 Sellers etal., 1985). The site of the regulatory phosphorylation is Ser-19 on the LC20 (Pearson et al., 1984). Myosin light chain kinase (MLCK) also phosphorylates Thr-18, albeit at a much lower rate (Ikebe et al., 1986). Thr-18 phosphorylation increases the actin-activated MgATPase activity (Ikebe et al., 1988), but does not increase the rate of actin filament sliding in either of the two motility assays (Sellers et al., 1985 Okagaki et al, 1991). 

Protein kinase C phosphorylates Ser-1 or Ser-2 and Thr-9 on the regulatory light chain (Bengur et al., 1987 Ikebe etal., 1987). This phosphorylation decreases the actin-activated MgATPase activity of smooth muscle myosin that was already phosphorylated by MLCK at Ser-19, by decreasing the apparent affinity for actin with no effect on the (Nishikawa et al., 1984). Protein kinase C phosphorylation of Ser-19 phosphorylated smooth muscle myosin does not affect the rate of movement in either of the two motility assays (Umemoto et al., 1989 Okagaki et al., 1991). Phosphorylation by protein kinase C in the absence of MLCK phosphorylation neither increases the actin-activated MgATPase activity nor supports in vitro motility (Sellers et al., 1985 Nishikawa et al., 1984). 

The fact that these additional phosphorylations can affect the actin-activated MgATPase assay and not the in vitro motility assays is not problematic, as the two processes are probably limited by different steps in the kinetic cycle. The kinetic step that limits the actin-activated MgATPase assay is thought to be phosphate release or some step preceding phosphate release (Sellers, 1985). The rate of in vitro motility is thought to be regulated by ADP release (see Chapter 1, this volume). 

Tropomyosin increases both the rate of in vitro motility and the actin-activated MgATPase activity (Umemoto and Sellers, 1990 Chacko and Eisenberg, 1990). The effect on the MgATPase activity is to increase the by a factor of two with no effect on the apparent affinity for actin (Umemoto and Sellers, 1990 Chacko and Eisenberg, 1990). The mechanism by which this occurs is not known, but the presence of tropomyosin on the thin filament must affect at least two different rate constants as discussed earlier, since it accelerates both the MgATPase activity and the rate of in vitro motility. 

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Johnson LN, Barford D The effect of phosphorylation on the structure and function of proteins. Annu Rev Biophys Bio-mol Struct 1993 22 199. 

Table 8.2 The effects of phosphorylation of tyrosine hydroxylase on enz3nne kinetics (based on Kaufman 1995)...

The biochemical mechanism of Mos action is not yet established. Mos has been found to phosphorylate cyclin B in vitro, and it is possible that this phosphorylation directly inhibits cyclin B proteolysis (Roy et al., 1990). However, such a direct effect of phosphorylation on cyclin B stability remains to be demonstrated, and it is alternatively possible that Mos inhibits (directly or indirectly) the proteolytic pathway responsible for cyclin B degradation. Mos has recently been found to stimulate mitogen-activated protein kinase (MAP kinase) in Xenopus oocytes,... 

Ramsey A., Fitzpatrick P. (1998). Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of catecholamines evidence for a novel regulatory mechanism. Biochemistry 37, 8980-6. 

Matts, R. L., Levin, D. H., and London, I. M. (1983). Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis. Proc. Natl. Acad. Sci. USA 80, 2559—2563. 

The major hormone-sensitive control point for the mobilization of fat and the (f-oxidation pathway is the effect of phosphorylation on the activity of the hormone-sensitive lipase of the adipose tissue. The major direct control point for (f oxidation is the inhibition of carnitine acyl-... 

A major way to control enzyme activity is the reversible phosphorylation of serine or threonine residues. It s such a major way to regulate enzymes that you will spend much of your time trying to remember whether or not enzyme X is activated or inactivated by phosphorylation, and you will invariably forget one or two of them on the exam. By remembering a couple of generalities,2 you can actually figure out a lot of the effects of phosphorylation on specific enzymes without really memorizing them. 

The functioning of G proteins may be influenced by phosphorylation. G proteins, as well as their associated receptors and RGS proteins, have been reported to undergo phosphorylation by a host of protein serine/ threonine kinases and protein tyrosine kinases. While the ramifications of receptor phosphorylation are becoming increasingly well understood (see Chs 23 and 24), the effect of phosphorylation of G proteins and RGS proteins, and its role in the regulation of physiological processes, have been more difficult to establish with certainty. This remains an important area of future investigation. 

The effect of phosphorylation at 13 /xmoles/g protein on functional properties was minimal. Both solubility and EAI of the phosphorylated protein were slightly higher, compared to those of intact protein (not shown). Phosphorylation with protein kinase from bovine cardiac muscle is restricted by the limited number of potential phosphorylation sites in soy proteins. Experiments are... 

E. Mameli found the effect of phosphoryl chloride on the f.p. of ehloroacetic acid indicates a mol. wt. less than normal at low cone, and, increasing with cone., exceeds the normal value with a 1-3 per cent. cone. 

In the medium and large neurofilament chains, where there are numerous K-S-P phosphorylation sites in the tail domains, the effect of phosphorylation is quite different. It has no visible effect on the state of filament assembly. It does, however, appear to be particularly important in determining axonal diameter (and concomitant conduction velocity), as well as transport properties and association with other cytoskeletal components. Experimentally, numerous phosphorylation sites have been shown to exist in a wide variety of IF proteins. Many others have been proposed on the basis of sequence motifs consistent with sites of known kinases. It has also been shown that mutations in which phosphorylation sites have been changed (see, for example, S35A in keratin 19) lead to various pathologies, including malformations in the filament assembly. 

Heeley, D. H., Watson, M. H., Mak, A. S., Dubord, P., and Smillie, L. B. (1989). Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle alpha-tropomyosin. / Biol. Chem. 264, 2424-2430. 

We also assessed the effect of phosphorylation of the ERp AF-1 in regulating interaction with SRC-1 in vivo using transfection experiments. Treatment of transfected cells with PD98059, a selective inhibitor of MAPK activation, completely abrogated the SRC-1-mediated activation of unliganded ER 3, whereas use of staurosporin, which inhibits protein kinase C, had no significant effect (Tremblay et al., 1999). The interaction between ER 3 and SRC-1 in the absence of hormone was also demonstrated in vivo and shown to be influenced by factors known to change the phosphorylation status of nuclear receptors. 

Huang, Y.T. and Kinsella, J.E. 1987. Effect of phosphorylation on emulsifying and foaming properties and digestibility of yeast protein. J. Food Sci. 52, 1684-1688. 

In this laboratory exercise, you will study the effects of phosphorylation and allosteric regulation on the activity of glycogen phosphorylase. In the first experiment, you will phosphorylate glycogen phosphorylase b in vitro using y-[32P]ATP and glycogen phosphorylase kinase. In the second experiment, you will study the effect of phosphorylation on glycogen phosphorylase activity, as well as the effect of AMP on glycogen phosphorylase b activity with the use of a coupled enzymatic (kinetic) assay (Fig. 15-3). 

RNA and DNA, and to study the effects of phosphorylation state and charge on the results. " In the polymer field. Berry et al. used BDE calculations for model alkyl urethanes to identify vulnerable sites to radical attack, and to examine the effect of branching on the photostability of urethane coatings. ... 

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