Protein phosphatases targeting subunits

Calcium-dependent regulation involves the calcium-calmodulin complex that activates smooth muscle MLCK, a monomer of approximately 135 kDa. Dephosphorylation is initiated by MLCP. MLCP is a complex of three proteins a 110-130 kDa myosin phosphatase targeting and regulatory subunit (MYPT1), a 37 kDa catalytic subunit (PP-1C) and a 20 kDa subunit of unknown function. In most cases, calcium-independent regulation of smooth muscle tone is achieved by inhibition of MLCP activity at constant calcium level inducing an increase in phospho-rMLC and contraction (Fig. 1). 

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. 

The phosphoprotein-ubiquitin ligases hgate ubiquitin exclusively to phosphorylated proteins. In this system, ubiquitination and degradation are controlled by the phosphorylation status of the target proteins, which is in turn dependent on the regulated activity of protein kinases (or protein phosphatases). Phosphorylation of the target proteins often occurs in sequence elements rich in the aminoacids P,E,S, and T (PEST sequences). For target proteins and the subunit structure, see 13.3.1. 

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. 

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.

The A kinase anchor proteins (AKAP), which are tightly associated with the cytoskeleton, serve as anchors for protein kinase A. In addition to the RII subimit of protein kinase A, the AKAP proteins also bring protein phosphatases and other protein kinases to the cytoskeleton in a targeted fashion. The AKAP79 protein binds protein kinase C and protein phosphatase 2B (calcineurin) as well as the RII subunit. The possibility to bring both a protein kinase and a protein phosphatase to the same place in the cell opens up the prospect of a coordinated and layered regulation of both enzyme activities. 

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). 

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

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. 

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)... 

The preparation of the C1-C21 subunit of the protein phosphatase inhibitor tautomycin was completed by J.A. Marshall et al., and it constituted a formal total synthesis of the natural product. The spiroketal carbon of the target was introduced by the Weinreb ketone synthesis between a lithioalkyne and A/-methoxy-A/-methylurea (a carbon monoxide equivalent). The triple bond of the resulting Weinreb s amide was first reduced under catalytic hydrogenation conditions to yield the corresponding saturated amide, which was reacted with another lithium acetylide to afford an ynone. 

Zhao XL, Gutierrez LM, Chang CF, Hosey MM. The a,-subunit of skeletal-muscle L-type Ca channels is the key target for regulation by a-kinase and protein phosphatase-lc. Biochem Biophysl Res Commun 1994 198 166-173. 

The myosin phosphatase targeting protein (MYPT) family a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phosphatase type Idelta. Arch Biochem Biophys 510 147-159... 

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... 

Dagda K, Zaucha JA, Wadzinski BE et al (2003) A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem 278 24976-24985... 

McCright B, Rivers AM, Audlin S et al (1996) The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 271 22081-22089... 

Davis AJ, Yan Z, Martinez B et al (2008) Protein phosphatase 2A is targeted to cell division control protein 6 by calcium-binding regulatory subunit. J Biol Chem 283 16104-16114... 

Xu Z, WUliams BRG (2000) The B56a regulatory subunit of protein phosphatase 2 A is a target for regulation by double-stranded RNA-dependent protein kinase PKR. Mol CeU Biol 20 5285-5299... 

Two systems have recently been described that result in methyl ester formation at the C-terminus of a protein. Both appear to be widely distributed in eucaryotic cells, but have not yet been observed in procaryotic cells. In one system, the C-terminal leucine residue of one or more 36 kDa cytosolic polypeptides is the target of methylation (Xie and Clarke, 1993). These methyl ester linkages are much more labile in cell extracts than would be predicted on the basis of their chemistry and this observation suggests the presence of a methylesterase activity (Xie and Clarke, 1994a). The protein substrate for this methyltransferase has recently been identified as the catalytic subunit of protein phosphatase 2A and its reversible methylation may modulate its activity (Xie and Clarke, 1994b). 

The intrinsic substrate specificity of protein phosphatases, when measured in vitro, is quite low. Much of the specificity of substrate dephosphorylation is achieved by targeting or scaffolding subunits that serve to localize the phosphatase in proximity to particular substrates, and also to reduce its activity towards other potential substrates. 

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. 

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