Protein kinase C phosphorylation

Pendergast, A. M., Traugh, J. A., and Witte, O. N. (1987). Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation. Mol. Cell. Biol. 7 4280-4289. 

Phorbol esters are promoters that interact with cellular receptors and activate protein kinase C. Usually protein kinase C is activated by Ca++ and diacylglycerol, both of which result from the hydrolysis of phosphoinositides catalyzed by phospholipase C. Phospholipase C is normally activated by several different growth factors. Thus phorbol esters bypass a tightly regulated step in the control of cell growth. Since protein kinase C phosphorylates various proteins, it is not known how this activity participates in establishing a cancerous line of cells. 

Chen et al. [62] have proposed that protein kinase C is involved in the fi receptor desensitization since activators of this enzyme potentate fi receptor uncoupling from the K+ channel. The fi receptor has consensus protein kinase C phosphorylation sites in its intracellular domains [8]. Arden et al. [88] have reported that morphine can induce the phosphorylation of the cloned fi receptor and Zhang et al. [64] have reported that activators of protein kinase C can induce the phosphorylation of the ft receptor. 

Gereau, R. W. and Heinemann, S. F. (1998) Role of protein kinase C phosphorylation in rapid desensitization of metabotropic glutamate receptor 5. Neuron 20,143-151. 

Nakajima, Y., Yamamoto, T Nakayama, T and Nakanishi, S. (1999) A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7. J. Biol. Chem. 274,27573-27577. 

H2A.1, H2A.2, and H2A.X are phosphorylated at serine residue 1 [8,9]. H2A.Z is not phosphorylated. In vitro protein kinase C phosphorylates H2A at serine residue 1 [10]. Telrahymena H2A is phosphorylated in the C-terminal sequence [11]. Tetrahymena H2A.1 is phosphorylated at serine residues 122, 124, and 129, while H2A.2 is modified at serine residues 122 and 128 (Fig. 3). Phosphorylation of H2A occurs in the transcriptionally active macronucleus of Tetrahymena thermophila, but not in the transcriptionally inert micronucleus [12]. Tetrahymena H2A variant hvl is phosphorylated [13]. H4, like H2A, is phosphorylated at N-terminal serine... 

The members of the protein kinase C family are central signal proteins and as such, are involved in the regulation of a multitude of cellular processes. A problem in the identification of substrates of protein kinase C is its low substrate specificity which often cannot be differentiated from that of protein kinase A, particularly in in vitro experiments. The consensus sequence of the phosphorylation sites in substrate proteins are similar to those of protein kinase A, in that basic amino acids are required in the neighborhood of the Ser/Thr residue to be phosphorylated. The following consensus sequences may be formulated for phosphorylation by protein kinase C ( = phosphorylation site) S /T XK/R K/RXXS /T K/RXXS /T XK/R K/RXS /T K/RXS /T XK/R (Pearson and Kemp, 1991). 

Leonard, A. S. and Hell, J. W. Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites, J. Biol. Chem. 1997, 272, 12107-12115. 

Orr GA, Han EK, Browne PC, et al. Identification of the major phosphorylation domain of murine mdrlb P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites. J Biol Chem 1993 268(33) 25054-25062. 

Wu X, et al. A critical protein kinase C phosphorylation site on the 5-HT(lA) receptor controlling coupling to N-type calcium channels. J Physiol 2002 538(Pt 1) 41—51. 

Belusa, R., Wang, Z--W., Matsubara, T., SahJgren, B-, Dulubova, J., Nairn, A. C-, Ruoslahti, E., Grecngard, E, and Aperia, A. (1997). Mutation of the protein kinase C phosphorylation site on rat nl Na, K -ATrase alters regulation of intracellular Na and pH and influences cell shape and adhesiveness. ]. Biul. Chem. 272, 20179-2CHS4. 

To this end, different ion fragmentation tools have been characterized with respect to phosphopeptide fragmentation, e.g., electron-capture dissociation (ECD) [31] and infrared multiphoton dissociation (IRMPD) [32]. An application of ECD in PTM analysis is the top-down protein characterization (Ch. 18.3.5) of carbonic anhydrase [33]. IRMPD is applied in the study on protein kinase C phosphorylation [30]. Both ECD and IRMPD were applied in a subsequent nano-ESI-FT-ICR-MS study on protein kinase A phosphorylation [34]. Combined ECD and IRMPD for multistage MS-MS in FT-ICR-MS was applied for phosphopeptide characterization [35]. ECD provides backbone cleavages (c- and z -ions) without H3PO4 loss, whereas in IRMPD the loss of H3PO4 is prominent and only a few backbone cleavages (b- andy-ions) are observed cf. Ch. 17.6.1). 

Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, et al. 1997. Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J. Biol. Chem. 272 5157-66... 

Numann R, CatteraU WA, Scheuer T 1991 Functional modulation of brain sodium channels by protein kinase C phosphorylation. Science 254 115—118... 

Direct phosphorylation of the glutamate transporter proteins themselves has only been reported for GLT and GLAST. Protein kinase C phosphorylates GLAST and thereby reduces the transport activity to 25% with no change in cell surface expression (Conradt and Stoffel, 1997). GLT was originally reported to be stimulated by phosphorylation of serine-113 (Casado et al., 1993), but a recent report (Tan et al., 1999) suggests that the protein kinase-C-mediated stimulation represents an effect of the expression system used rather than an effect on GLT. Further studies are required to sort out the controversy. 

Another type of myristoyl switch has been reported for the MRACKS proteins which are substrates of protein kinase C (see Section 7.4). The membrane binding of the MARCKS proteins is mediated by myristate plus basic motif. Protein kinase C phosphorylation within the basic motif introduces negative charges into the positively charged region. This reduces the electrostatic interactions with the acidic phospholipids and results in displacement of the MARCKS proteins from the membrane and into the cytosol. 

PKC Protein kinase C Phosphorylation, Ser 378 Takenaka etal. (1995) Baudier et al. (1992)... 

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). 

After activation, protein kinase C phosphorylates specific serine and threonine residues in target proteins. As with cAMP-stimulated protein kinase, the specific cellular responses to protein kinase C activation depend on the ensemble of target proteins that become phosphorylated in a given cell. Known target proteins include calmodulin, the insulin receptor, / -adrenergic receptor, glucose transporter, HMG-CoA reductase, cytochrome P-450, and tyrosine hydroxylase. 

Consensus sites for phosphorylation were evident in the neuronal NOS enzyme from the predicted protein sequences derived from cDNA analysis. In vitro biochemical studies indicate that nNOS can be phosphorylated by calcium/calmodulin-dependent protein kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. Phosphorylation of nNOS by all of these enzymes decreases NOS catalytic activity in vitro (Dawson and Snyder, 1994 Bredt etal., 1992 Dinerman etal., 1994a). Calcineurin, a protein phosphatase, dephosphorylates NOS and subsequently increases its catalytic activity (T. M. Dawson etal., 1993). Multiple levels of constitutive nNOS regulation are thus possible by phosphorylation. 

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