Type 1 Catalytic Subunit

Several laboratories have demonstrated the existence of PPlc isoforms. (Dombradief a/., 1990 Sasaki ef al., 1990 Wadzinski et al., 1990). We will use the no- 

FIGURE 1 Comparison of the sequences of the isoforms of the PPl catalytic subunit (Sasaki eial., 1990). Identical residues are indicated by dots lack of aligning residues is indicated by bar. 

The PPlc8 isoform is associated with the trimeric phosphatase from chicken and turkey gizzard (Okubo et al., 1993). Alessi et al. (1992) also showed the presence of this isoform (in their nomenclature it is the P-isoform). The same isoform is associated with skeletal muscle myosin (Dent etal., 1992). It is reasonable to expect that each isoform has distinctive properties, although this has not yet been demonstrated. Zhang et al. (1992) developed an expression system for the PPlc subunit and found that the four isoforms were similar with respect to inhibition by 12 and okadaic acid (Zhang et al., 1993). They required Mn2+ for full activity. The PPlc has also been expressed in the baculo- 

The PPlc8 isoform is present in smooth muscles and was detected bound to gizzard myosin (Okubo et al., 1994). In addition, cDNA clones for PPlc8 have been isolated from cDNA libraries of chicken gizzard 

Consideration of other catalytic subunits (PP2A, etc.) is beyond the scope of this article and the reader is referred to Cohen (1991). 

---

Durfee T, Becherer K, Chen PL et al (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev 7 555-569... 

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

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. 

Information about the putative folding of the H,K-ATPase catalytic subunit through the membrane has been obtained by the combined use of hydropathy analysis according to the criteria of Kyte and Doolittle [51], identification of sites sensitive to chemical modification [46,48,50,52-55], and localization of epitopes of monoclonal antibodies [56]. The model of the H,K-ATPase catalytic subunit (Fig. 1) which has emerged from these studies shows ten transmembrane segments and contains cytosolic N- and C-termini [53]. This secondary structure of the catalytic subunit is probably a common feature of the catalytic subunits of P-type ATPases, since evidence supporting a ten a-helical model with cytosolic N- and C-termini has also been published recently for both Ca-ATPase of the sarcoplasmic reticulum and Na,K-ATPase [57-59]. 

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... 

The uniquely high resolution structural data available for the SERCAIa Ca2+ pump illuminates the structure of all P-type transporters. Unlike the Na,K pump, the catalytic subunit of the SERCA Ca2+ pumps is active and does not require association with another subunit. However, the cardiac isoform, SERCA-2a, associates with a small membrane protein, phospholamban, that can... 

Class I B catalytic subunits (110 kDa) are stimulated by Py subunits derived from G proteins (see Ch. 19). A regulatory protein (plOl) that associates tightly with the catalytic subunit has been isolated. Both type A and type B catalytic subunits of class I PI3K interact with a small... 

How do a wide variety of neurotransmitters and hormones produce tissue- and cell-specific biological responses, if many such responses are mediated by the same intracellular messengers, cAMP and cAMP-depen-dent protein kinase Specificity is achieved at two levels at the level of tissue-specific receptors for the neurotransmitter or hormone and at the level of tissue-specific substrate proteins for the protein kinase. Only tissues that possess specific receptors will respond to a certain neurotransmitter or hormone. Moreover, since all cells contain very similar catalytic subunits of protein kinase A (see Ch. 23), the nature of the proteins that are phosphor-ylated in a given tissue depends on the types and amounts of proteins expressed in that tissue and on their accessibility to the protein kinase. 

The cytochrome he complex in eukaryotes is a homodimeric, multi-subunit entity (Figure 13.13a). Each monomer has three catalytic subunits a cytochrome b, with two b-type haems, one Rieske ISP containing a [2Fe-2S] cluster and one cytochrome c, with... 

Akt activity is induced in a PI-3K-dependent manner immediately suggesting that the phosphorylated lipid products of PI-3K mediate the activation. Incubation of purified Akt with purified 3-phosphorylated phospholipids results in various extents of activation (44,46). These lipids, such as PtdlnsJP, PtdIns(3,4)P2> and PtdIns(3,4,5)P3, specifically associate with PH domains in a number of proteins (47). Furthermore, co-transfection of a dominant-negative form of PI-3K (delta-p85) also inhibits Akt activation (43). It was later shown that introduction of constitutively active mutants of the catalytic subunit of PI-3K was sufficient to activate Akt in cells (46,48). These studies strongly implicate Akt as a downstream effector of growth-factor-stimulated PI-3K activation in a variety of cell types. 

The actions of cAMP occur almost exclusively via regulation of cAMP-de-pendent protein kinase (PKA), which consists of catalytic and regulatory subunits (Nestler and Duman 1999). There are three different isoforms of the catalytic subunit and four isoforms of the regulatory subunit. In the inactive state, in the absence of cAMP, PKA exists as a dimer of two catalytic and two regulatory subunits. Upon binding of cAMP to the regulatory subunits, the catalytic subunits are released and can phosphorylate substrate proteins. The types of cellular proteins that serve as substrates for PKA include metabohc enzymes, receptors, ion channels, effector proteins, and gene transcription factors. 

Autonomic receptors further regulate calcium influx through the sarcolemma (Fig. 15.1). (3-Adrenergic stimulation results in the association of a catalytic subunit of a G protein coupled to the (3-receptor. This stimulates the enzyme adenylyl cyclase to convert ATP to cyclic adenosine monophosphate (cAMP). Increasing cAMP production results in a cAMP-dependent phosphorylation of the L-type calcium channel and a subsequent increase in the probability of the open state of the channel. This translates to an increase in transsarcolemmal calcium influx during phase 2 (the plateau phase) of the cardiac muscle action potential. The effects of transient increases in intracellular levels of cAMP are tightly con-... 

There are two prominent types of mammalian cAMP-dependent protein kinases.51 61 The catalytic subunit is identical for both the 41-kDa peptide as isolated from beef heart has 350 residues and an N terminus blocked by a myristoyl (tetradecanoyl) group.62 One phosphoserine and one phosphothreonine are also present.51 The 50-kDa regulatory subunits vary in size and may also be subject to additional regulation by phosphorylation.63 Three-dimensional structures are known for both the catalytic62 64 65 and the regulatory66 subunits. A cyclic GMP (cGMP)-activated protein... 

It is the world s most abundant enzyme and probably the most abundant protein. The enzyme found in plants has eight copies each of two types of subunits. The larger subunit contains the catalytic site the role of the smaller subunit is unclear. 

E. coli uses nitrate as a terminal electron acceptor through a respiratory, dissimilatory nitrate reductase whose synthesis is induced when nitrate is provided, and which is repressed by oxygen. Nitrate reductase is discussed with other molybdoenzymes in Section 62.1.9, and catalyzes the reduction of nitrate to nitrite. The enzyme is isolated from the cytoplasmic membrane of E. coli, and contains three subunits (a, j8 and y) although the y-subunit may be absent in some preparations. The -y-subunit is a b-type cytochrome, and the a-subunit is reported to be the catalytic subunit. The enzyme contains a number of iron-sulfur clusters, including a HiPIP and at least two ferredoxins.1054,1437... 

Figure 4.2 Crystal structure of potato tuber ADP-glucose small (catalytic) subunit monomer. The catalytic domain is in yellow and the beta-helix domain is in pink. ADPGlc is shown in atom type carbon atoms are green, oxygen atoms are red, nitrogen atoms are blue, phosphorus atoms are magenta, and the sulfate group is orange. (The full color version ofthis figure can be found atwww.Elsevier.books.com/)...

Quantitative evaluation of the strength of interactions between subunits is often difficult because of the multiplicity of the interactions. However, subunit exchange experiments can provide information about the relative interaction strength. In the study on aspartate transcarbamoylase,24-25 the dissociation rates of mutant subunits were determined by subunit exchange leading to hybrid molecules. The formation of hybrid molecules composed of native and succinylated catalytic subunits could be easily monitored by polyacrylamide gel electrophoresis, and it was suggested that the trimeric structure of a mutant in which Gly-128 was replaced by Asp was much more stable than that of the wild-type.24 25 ... 

---