Phosphorylation sites

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

FIGURE 25.4 Models of the acetyl-CoA carboxylase polypeptide, with phosphorylation sites indicated, along with the protein kinases responsible. Phosphorylation at Ser " is primarily responsible for decreasing the affinity for citrate. 

Histone phosphorylation is a common posttranslational modification fond in histones, primarily on the N-terminal tails. Phosphorylation sites include serine and threonine residues, tyrosine phosphorylation has not been observed so far. Some phosphorylation events occur locally whereas others occur globally throughout all chromosomes during specific events like mitosis. Histone phosphorylation is catalyzed by kinases. Removal of the phosphoryl groups is catalyzed by phosphatases. 

Protein Kinase C. Figure 1 Domain structure of PKC family members showing regulatory modules (pseudosubstrate sequence and C1, C2, and PB1 domains) and the kinase core. Shown below are the structures of the C1 domain of PKC 5 with bound phorbol (purple), the C2 domain of PKC (3 with bound Ca2+ (pink spheres), and the recently solved structure of the kinase domain by Grant and coworkers [1] of PKC pil with phosphorylation sites indicated in pink. Figure adapted from Newton (2003). 

When 14-3-3s were first identified as phosphorylation dependent binding proteins (note that a selection of non-phosphorylated targets are known), target protein phosphorylation sites were mapped and it was immediately apparent that 14-3-3s bound preferentially to specific phosphorylation motifs. The advent of oriented... 

HSFl phosphorylation must be sensitive to nonheat inducers of HSF-DNA binding activity because HSFl phosphorylation can be achieved at 37 °C by other inducers of the HS response. HSF 1 contains polypeptide sequences that could serve as substrates for well characterized protein kinases, but few of these are known to be heat inducible. One family of protein kinases, the S6 protein kinases, have already been shown to exhibit heat inducible activity however, their peak level of activity during HS occurs well after the maximal induction of HSF phosphorylation (Jurivich et al., 1991). Thus, other protein kinases are likely to be directly linked to the phosphorylation of HSF. Some of the putative protein phosphorylation sites on HSF include motifs for protein kinase C, casein kinase, and enterokinase. There are tyrosine sequences that match substrates for known tyrosine kinases, but whether these residues are accessible to phosphorylation is not established. 

Upon activation by CXCLl2, CXCR4 is quickly phosphorylated and internalized. Several residues in its C-terminus tail have been identified as potential phosphorylation sites by truncation and mutagenesis studies as reviewed in Busillo and Benovic (2007). Removal of 45 amino acid residues from the C-terminal of CXCR4 led to elimination of CXCL12-induced phoshorylation, enhanced receptor activity... 

Huttenrauch F, Nitzki A, Lin FT, Honing S, Oppermann M (2002) Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif. J Biol Chem 277 30769-30777 Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD, Frenkel D, Li J, Sidman RL, Walsh CA, Snyder EY, Khoury SJ (2004) Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor lalpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A 101 18117-18122... 

A sequence of ten amino acids (ICS-D-KTGTLT) around the phosphorylation site of Na,K-ATPase (Asp ) is highly conserved among the Na,K-, H,K-, Ca-, and Id-pumps [6]. There is also homology with the subunit of FpATP synthetase of mitochondria and chloroplasts (see [6]) except that Asp is replaced by Thr. Accordingly a covalent phosphorylated intermediate is not formed in Fi-ATPase. Mutagenesis of the phosphorylated aspartate residue in Na,K-ATPase [82], Ca-ATPase [87], or H-ATPase [88] completely blocks activity. 

Hydropathy analysis predicted that there are four major transmembrane domains (M1-M4) prior to the phosphorylation site at Asp . The existence of these four transmembrane segments in the N-terminal half of the catalytic subunit is generally accepted for all P-type ATPases. The four transmembrane sequences are followed by a large cytosolic loop that contains the phosphorylation site Asp, the pyridoxal... 

It has been established by substitution of for Mg that, prior to phosphorylation, the divalent cation binds at a cytosolic site with a stoichiometry of about 1 mol per phosphorylation site [124,125]. These experiments also demonstrated that the phosphorylation rate is sensitive to the nature of the divalent cation bound. With Mg bound, the phosphorylation rate is about 20 times faster than with Ca bound. The divalent cation dissociates after dephosphorylation, suggesting that it is tightly bound to the phosphoenzyme during the reaction cycle. It was also demonstrated that the type of divalent cation that occupies the divalent cation site required for phosphorylation is important for the step 2K E2-P to 2K E2 P to 2K E2 [124,125]. With Mg bound, the 2K E2-P conformer is -sensitive, whereas with Ca bound, the intermediate is -insensitive. 

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