Targeting Subunits

Using the monoclonal antibody to the 58-kDa fragment, it was found, by Western blots, that a protein similar or identical to the 130-kDa subunit was present in a wide range of tissues (Okubo et al., 1994). A crossreacting species was present in each of the smooth muscles examined (stomach, gizzard, aorta, oviduct, and small intestine) and was also detected in brain, spleen, kidney, lung, and cardiac muscle. Slightly different apparent molecular masses were observed in many tissues, for example, in cardiac muscle a doublet of 143 and 137 kDa was observed. It is not known if these different immunoreactive species are different isoforms. Northern blots confirmed these data, and 

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Allen, P. B., Kwon, Y. G., Naim, A. C. and Greengard, P. Isolation and characterization of PNUTS, a putative protein phosphatase 1 nuclear targeting subunit. J. Biol. Chem. 273 4089-4095,1998. 

NSF N-ethylmaleimide-sensitive factor PNUTS phosphatase 1 nuclear targeting subunit... 

Skpl serves as an adaptor protein that provides a molecular link between Cull/ Rod and the F-box proteins [4, 5]. The Skpl protein contains two separate protein-interaction domains that are conserved among its family members between species [21]. The N-terminal region of Skpl (- l-70 a.a.) interacts with Cull while the C-terminal half (100-163 a.a.) binds the F-box proteins [21]. The use of Skpl as an adaptor to link the core ubiquitin E3 ligase components of Cull/Rocl with numerous and diverse substrate-targeting subunits, the F-box proteins, represents a strategy to specifically target many proteins for ubiquitination... 

The role of Skpl is to bring the substrate-targeting subunit, the F-box protein, into proximity with the Cull/Rocl/E2 complex to promote ubiquitin transfer from the E2-ubiquitin to the F-box protein-bound substrates. 

F-box proteins serve as the substrate-targeting subunit of the SCF ubiquitin E3 ligase [5]. They are structurally diverse but they all contain a relatively conserved signature motif of about 45-50 amino acids [5]. This motif, the F-box, was initially... 

Stepanova, L., X. Leng, S. B. Parker, and J. W. Harper. Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4. Genes Dev. 10 1491-502.1996. 

The Ser/Thr phosphatases exist mostly as heterodimers composed of a catalytic subunit and another subunit to which a specific function for localization of protein phosphatases is often attributed. In the case of protein phosphatase I, this subunit is known as the targeting subunit, its function is described in more detail in 7.7. 

Organizing glucose disposal emerging roles of the glycogen targeting subunits of protein phosphatase-1. Diabetes 49, 1967-1977. 

When neither tissue nor cell lines are available, another alternative is purified target antigen. This type of control might be necessary when the test article is directed against a bacterial toxin and there is no cell line available that expresses the whole toxin or the targeted subunit. In these cases the negative tissue control would consist of a protein that is known to be nonreactive with the antibody being tested. 

Regulation by relocation. Mechanisms to overcome cellular compartmentalization are pivotal points of regulatory control. Relocation may involve targeting proteins. Targeting subunits guide both protein kinases and phosphatases to their substrates, located in different subcellular compartments. 

Comparing the structures in Fig. 7.2 and Plate 13 reveals how highly conserved the catalytic domains of Ser/Thr phosphatases are. Therefore their functional diversity must be due to different regulatory and targeting subunits that direct the phosphatase to discrete subcellular locations (Fig. 7.3). 

Rg. 7.3 The targeting subunit Is a discrete entity, different from the substrate. 

The role of targeting subunits was first recognized in the case of protein kinase C, as it became clear that isotbrms of protein kinase C and their substrates have different subcellular locations, raising the question of how the enzymes find their substrates. PKCs are membrane-attached Ser/Thr protein kinases. They were discovered by... 

J. Wu, J. Liu, I. Thompson, C.J. Oliver, S. Shenolikar, and D.L. Brautigan. 1998. A conserved domain for glycogen binding in protein phosphatase-1 targeting subunits FEBSLett. 439 185-191. (PuhMed)... 

It is not known what prevents the dephosphorylation of Inhibitor by PPl. Inhibitor-1 has also been found to act as an inhibitory subunit of a trimeric complex with PPl and the eIF2a-targeting subunit GADD34 [38],... 

Turowski P, Myles T, Hemmings BA et al (1999) Vimentin dephosphorylation by protein phosphatase 2A is modulated by the targeting subunit B55. Mol Biol Cell 10 1997-2015... 

Sphere and Dumbell Dimer. Dumbell dimers reacting with a spherical target represent the simplest case of structured reactants. The model used is depicted in Figure 2. The radii of the target (subunit 1) and dimer (subunits 2 and 3) were 2.0 and 0.5 A, respectively. The target sphere and either one or both dimer subunits were taken to be reactive. The criteria for a reactive collision were R < 3 A, and (for the cases with only one dimer subunit reactive) 0 < 90°. To study the effects of direct forces on reaction rates, variable charges (Q ) were placed at the centers of the subunits. 

PPI is a major dass of eukaryotic Ser/Thr-specific protein phosphatases that regulate diverse cellular processes such as cell cycle progression, muscle contraction, carbohydrate metabolism, protein synthesis, transcription, and neuronal signaling. Its action is modulated and regulated by assodation with subunits induding various inhibitor proteins and multiple targeting subunits of which nearly 30 proteins have now been identified (review Aggen et al., 2000). The activity of the inhibitory proteins can be controlled via phosphorylation by protein kinase A as outlined in Fig. 7.16. 

Of the many targeting subunits of PPI, the skeletal musde glycogen binding subunit Gm has been best studied and will be presented below in more detail. 

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