Protein phosphatase Localization

Allen, P. B., Ouimet, C. C. and Greengard, P. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc. Natl Acad. Sci. U.S.A. 94 9956-9961, 1997. 

Fig. 1.36. Regulation of the sub-ceUular localization of the transcription factor SWI5 in yeast by phosphorylation. The subcellular localization of the SWI5 protein is regulated by phosphorylation/ dephosphorylation. In the phos-phorylated state, SWI5 is found in the cytoplasm, while in the under-phosphorylated state it is localized in the nucleus. Phosphorylation and dephosphorylation are catalyzed by either protein kinases or protein phosphatases and can be controlled via signal transduction chains.

The phosphorylation state of the transcription factor NF-AT has a different effect on translocation. The phosphorylated form of this protein is localized in the cytosol and requires dephosphorylation by the protein phosphatase calcineiuln in order to be translocated to the nucleus (see also 7.5.2). Other examples for phosphorylation-dependent nuclear translocation include the STAT-proteins (see 11.1.3.2) and the SMAD-proteins (see 12.1.2). 

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. 

With the help of the localization subrmit, a protein phosphatase can be directed to distinct subcellular locations at which the substrates of the protein phosphatase are also... 

The extent and specificity of the reactions of protein kinases and protein phosphatases are extremely dependent on the degree to which substrate and enzyme are localized at the same place in the cell. Many substrates of protein kinases occur either as membrane associated or particle associated forms (see 7.6.1, enzymes of glycogen metabolism). For protein kinases or protein phosphatases to perform their physiological function in a signal transduction process, they must be transported to the location of then-substrate in many cases (review Hubbard and Cohen, 1992 Mochly-Rosen, 1995). This is vahd both for the Ser/Tbr-specific protein kinases as well as for many Tyr-speci-fic protein kinases. In the course of activation of signal transduction pathways, com-partmentahzation of protein kinases, redistributed to new subcellular locations, is often observed. 

The principle of targeted localization is shown in Fig. 7.22. In addition to the binding site for the corresponding protein kinase (or protein phosphatase), the localization subunit also has a specific binding site for an anchor protein, found at a subceUular site in the region where protein phosphorylation should take place. Through the interaction of anchor protein and localization subunit, the catalytic subunit is fixed at the desired location and is able to preferentially convert substrate localized at the same location. 

Fig. 7.22. The prindple of targeted localization of protein kinases and protein phosphatases. The spatial configuration between the catalytic subunit of a protein kinase or protein phosphatase and a membrane-associated substrate is mediated by localization subunits that specifically bind to membrane-localized anchor proteins. The specificity of co-localization is predominantly achieved at the level of binding of the localization subunit to the anchor protein. The co-localization is regulated, in particular, by the interaction of the catalytic subunit with the localization subunit. In the membrane-associated form, the catalytic subunit has increased activity towards membrane-bound substrates.

By bringing together various protein kinases and protein phosphatases, anchoring proteins organize signal transduction events and can create localized and efficient signal events at specific subcellular sites. 

The G subimit of protein phosphatase I, occurring in the glycogen-boimd form, is considered as its localization subimit. The G subunit enables targeted localization of the catalytic subunit of protein phosphatase I to glycogen so that a close spatial orientation of protein phosphatase and its substrates, the enzymes of glycogen metabolism, is created (see 7.6.2). 

Hie subcdlular localization of the kinase or phosphatase also plays a crucial role for the activity of protein kinases and protein phosphatases. Many physiological functions of protein kinases and protein phosphatases depend on the enzyme being brought, with the help of specific protein-protein interactions, to certain subceUular locations in the vicinity of its substrate. 

Fig. 1. Double label immunohistochemistry on rai liver. An acetone-fixed rat liver section was incubated with a polyclonal antiserum raised in rabbit to a hepa-tocyte cell surface protein (courtesy of Dr. S. Stamatoglou) and a mouse monoclonal antibody against a bile duct specific cytokeratin (courtesy of Dr. E. B. Lane). The hepatocyte protein was localized by use of a secondary peroxidase-conjugated antibody resulting in a red/brown product (thin arrow). The bile duct cytokeratin was identified by using an alkaline phosphatase-conjugated secondary antibody giving a blue color (thick arrow).

Usuda, N., Arai, H., Sasaki, H., Hanai, T., Nagat, T., Muramatsu, T., Kincaid, R. L., and Higuchi, S. 1996. Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin dependent protein phosphatase (calcineurin) in central nervous system neurons Immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. J. Histochem. Cytochem. 44 13-18. 

Our laboratory has provided significant contributions in the area of myristoyla-tion. We discovered and purified the myristoyl-CoA binding protein (MCBP) from bovine cardiac muscle (Raju and Sharma 1997). In cardiac tissues there is a high level of cAMP-dependent protein kinase expression whose catalytic subunit is myristoylated (Carr et al. 1982). The catalytic subunit of cAMP-dependent protein kinase and the beta subunit of calcineurin are myristoylated proteins localized in the cytoplasm (Selvakumar et al. 2006, 2002 Rajala et al. 2000 Johnson et al. 1994 Carr et al. 1982 Aitken et al. 1982). Recently it has been shown that dephosphorylation of the catalytic subunit of myristoylated and nonmyristoylated cAMP-dependent protein kinase at Thr-197 by cellular protein phosphatase and protein... 

I. Konietzka, P. Gres, G. Heusch, R. Schulz, Co-localization of connexin 43 (Cx43) and protein phosphatases in preconditioned myocardium in pigs, J Mo Cell Cardiol 36,757 (Abstract) (2004). 

Both mPA-PLAjU and mPA-PLAj/ , like many phosphate-recognizing enzymes such as phosphatase and ATPase [40], are inhibited by vanadate [51]. When expressed in insect Sf9 cells, neither mPA-PLAja nor mPA-PLAj/ were secreted. Both were recovered from the Triton X-lOO-insoluble fraction of the cell membrane. An immunofluorescence analysis confirmed that these proteins are localized exclusively to the plasma membrane. Both mPA-PLAja and -fi may locahze in the detergent-resistant membrane domain, referred to as raft structures, and function as lysoPA-producers under certain conditions [51]. 

Helps NR, Luo X, Barker HM et al (2000) NIMA-related kinase 2 (Nek2), a cell-cycle-regulated protein kinase localized to centro-somes, is complexed to protein phosphatase 1. Biochem J 349 509-518... 

Strack S, Zaucha JA, Ebner EF et al (1998) Brain protein phosphatase 2A developmental regulation and distinct cellular and subceUular localization by B subunits. J Comp Neurol 392 515-527... 

Gigena MS, Ito A, Nojima H et al (2005) A B56 regulatory subimit of protein phosphatase 2A localizes to nuclear speckles in cardio-myocytes. Am J Physiol Heart Circ Physiol 289 H285-H294... 

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