Sites of phosphorylation

FIGURE 10.10 The reaction of tridated sodium borohydride with the aspartyl phosphate at the active site of Na, K -ATPase. Acid hydrolysis of the enzyme following phosphorylation and sodium borohydride treatment yields a tripeptide containing serine, homoserine (derived from the aspartyl-phosphate), and lysine as shown. The site of phosphorylation is Asp" in the large cytoplasmic domain of the ATPase. 

Protein kinase A (PKA) is a cyclic AMP-dependent protein kinase, a member of a family of protein kinases that are activated by binding of cAMP to their two regulatory subunits, which results in the release of two active catalytic subunits. Targets of PKA include L-type calcium channels (the relevant subunit and site of phosphorylation is still uncertain), phospholam-ban (the regulator of the sarcoplasmic calcium ATPase, SERCA) and key enzymes of glucose and lipid metabolism. 

Two principal methods are available (1) western blotting using phos-phospecific antisera and (2) isoelectric focusing followed by western blotting. Protocols based on mass spectroscopic methods may also be valuable, especially where either the phosphorylation site has not been identified or the protein contains multiple sites of phosphorylation. 

During the electron tranters at the three classic sites of phosphorylation (marked I, II, and III), protons are pumped out of the mitochondria into the cytoplasm. The exact number of protons pumped at each site is somewhat controversial however, this proton pumping makes the interior of the mitochondria alkaline. 

FIGURE 21-6 Schematic illustration of the overall structure and regulatory sites of eleven different phosphodiesterase subtypes. The catalytic domain of the phosphodiesterases are relatively conserved, and the preferred substrate(s) for each type is shown. The regulatory domains are more variable and contain the sites for binding of Ca2+/calmodulin (CaM) and cGMP, as well as GAF and PAS domains. The regulatory domains also contain sites of phosphorylation by cAMP-dependent protein kinase (PKA). 

FIGURE 23-5 Schematic diagram of tyrosine hydroxylase with sites of phosphorylation indicated (yellow) along with the protein kinases. 

Histone phosphorylation was first reported in 1966 [1,2]. The four core histones, histone variants, and HI histones are phosphorylated, with the sites of phosphorylation being found in both the amino-terminal and carboxy-terminal portions of the histones [3] (Figs. 1 and 2). Phosphorylation of the core histones has been implicated in transcription, replication, chromosome condensation, and DNA repair. 

Fig. 2. Histone HI modifications. Sites of phosphorylation (P) and ADP ribosylation (step ladder) on mouse Hl -3 are shown.

Aromatic amino acids are biogenetic precursors of neuroamines (dopamine, serotonin, histamine, etc.). On the other hand, phenylalanine (Phe) is frequently present in peptide sequences, while tyrosine is an important site of phosphorylation of proteins. Aromatic amino acids and neuroamines fluorinated on the aromatic ring have been the focus of many investigations. Indeed, after incorporation in polypeptides and proteins, they can be used as probes in NMR and in PET. 

Figure 4.8 Schematic structure of the mammahan P -adrenergic receptor. There are seven memhrane-spanning helical regions composed of hydrophobic amino acid sequences, and at least two glutamine-hnked glycosylation sites near the N-terminal. P shows potential sites of phosphorylation hy cAMP-linked protein kinase arrows indicate serine and threonine molecules that can he the site of regnlatory phosphorylation hy receptor kinase.

Additional information <1, 5, 6> (<5> 50 kDa C-terminus-truncated mutant RK lacking the last 59 amino acid.s shows abolished light-dependent translocation and is unable to phosphorylate photoactivated rhodopsin, but phos-phorylates the small peptide substrate RRREEEEESAAA like wild-type RK [16] <6> RK knockout mice [18,36] <1> mutations at the autophosphorylation region affect the Km for ATP and change the initial site of phosphorylation on photolyzed rhodopsin, influence of mutations on the affinity for he-parin-Sepharose [33] <5> GRKl mutations causing the Oguchi disease [36]) [16, 18, 33, 36]... 

S ATP -I- myosin I heavy chain <1, 2, 9-11> (<10> major site of phosphorylation is Ser8 [22] <2> 35 kDa trypsin fragment of the C-terminus of the maximally activated, phosphorylated enzyme is fully catalytically active and contains 2 thirds of the autophosphorylation sites of the native enzyme [20] <9,10> substrate myosin ID [19,22] <2> higher activity with membrane-bound substrate myosin I [17] <2> substrates are heavy chains of myosin lA and IB [6,7,17] <2,11> substrate is heavy chain of myosin IC [7,23,24] <2> a basic amino acid is essential on amino-terminal side of phosphorylation site, two are preferable, and a Tyr-residue is essential two residues away on the COOH-terminal side [7] <2> contains two myosin heavy chain kinases one for myosin I and one for myosin II... 

Waygood, E.B. Enzyme I of the phosphoenolpyruvate sugar phosphotransferase system has two sites of phosphorylation per dimer. Biochemistry, 25, 4085-4090 (1986)... 

Chance and associates employed spectrophotometry on intact mitochondria or submitochondrial particles to investigate both the sequence of carriers and the sites of phosphorylation. Using the dual wavelength spectrophotometer, the light absorption at the absorption maximum (Aniax) of a particular component was followed relative to the absorption at some other reference wavelength (Aref). The principal wavelengths used are given in Table 18-6. From these measurements the state of oxidation or reduction of each one of the carriers could be observed in the various states and in the presence of inhibitors. The... 

ATP is coupled to the electron transport to cytochrome c. Thus, we have experimental evidence that when one-electron carriers such as the cytochromes are involved, the passage of two electrons is required to synthesize one molecule of ATP. Furthermore, from experiments of this type it was concluded that the sites of phosphorylation were localized in or related to complexes I, III, and IV. 

A small fraction of histone H2A undergoes phosphorylation and dephosphorylation continuously,43 but HI and H3 are phosphorylated and dephosphor-ylated at specific stages of the cell cycle. Phosphorylation of HI has been thought essential for "condensation" of chromatin,44 45 the folding into the tightly packed chromosome structures. However, more recent experiments point to the N terminus of histone H2B as the required site of phosphorylation for chro-... 

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