Background Information

A key feature of protein phosphorylation (as compared to other protein modifications, such as proteolysis) is its reversibility. The ability to phosphorylate a substrate in vitro requires that the kinase responsible for phosphorylation be known. However, many cellular protein kinases are themselves activated via protein phosphorylation, making it difficult to use them in vitro to phosphorylate the substrate. Establishing the physiological relevance of many in vitro phos-phorylations is also made difficult because these studies often do not take account of the physiological concentrations of the kinase and its substrate. In vitro experiments also tend to ignore the importance of the subcellular local ization of the substrate and the kinase, sometimes leading to modifications that do not occur in vivo. For these reasons, knowledge of the physiological stimuli that regulate the substrate's function can be used to promote substrate phosphorylation as well as to provide important clues in the identification of protein kinases that phosphorylate the substrate.

Most cellular phosphoproteins are subject to covalent modification at multiple sites by more than one protein kinase. Therefore, in vitro studies with purified protein kinases often fail to recreate the spectrum of phosphoryla-tions seen in the intact cell, and this also makes it difficult to define the role of individual cova-lent modifications. Thus, a new strategy of

Post-Translational Modification: Phosphorylation and Phosphatases

Detection of Phosphorylation by Enzymatics Technique analyzing the role of multisite phosphorylation has been developed using proteins that have been modified in the intact cell. However, it is particularly important to isolate such substrates in the presence of phosphatase inhibitors and to preserve the protein-bound phosphates during lengthy purification procedures.

Biochemical studies of protein phos-phatases have established the in vitro substrate specificity of these enzymes, which distinguish between different phosphoamino acids (phos-phoserine/phosphothreonine versus phospho-tyrosine) and different phosphoprotein substrates. These enzymes can also show preferential dephosphorylation of specific sites (Shenolikar and Nairn, 1991; Charbonneau and Tonks, 1992; Shenolikar, 1994). In vitro dephosphorylation by selected phosphatases may not only define the functional role of selected covalent modifications but also identify "silent" or "structural" phosphorylations that do not directly regulate the protein function. These phosphorylations may play an indirect role in modulating protein phosphorylation or dephos-phorylation of other sites.

This unit has focused on commercially available phosphatases that have been used to characterize the functional importance of protein phosphorylation. For example, several growth-associated kinases, including the mito-gen-activated protein kinases (MAP kinases) and the cyclin-dependent kinases such as cdc2, are phosphorylated on adjacent threonine and tyrosine residues. Phosphorylation of these enzymes is controlled by kinases and phosphatases that are themselves highly regulated in the intact cell. In vitro dephosphorylation by serine/threonine-specific or tyrosine-specific phosphatases both established the functional importance for phospho-rylations even prior to the identification of the kinases and phosphatases that regulate these proteins.

The most direct method for detecting protein phosphorylation is clearly metabolic labeling of cells with 32Pi (see unit 13.2) and subsequent isolation of the 32P-labeled substrate protein. However, it is often difficult to develop a rapid purification procedure for the specific protein of interest without selective reagents such as monospecific antibodies or other affinity li-gands. Significant labeling of proteins is also a problem given the low abundance of many regulatory proteins and the slow turnover of their protein-bound phosphate under "basal" conditions. In cases where [32P]phosphate can be readily incorporated into the protein, in vitro dephosphorylation by selected phosphatases can establish the functional importance of the modification. Ability to correlate loss of [32P]phosphate from a specific polypeptide with a functional change provides the best evidence for the physiological importance of protein phosphorylation.

Initial experiments should use a general phosphatase such as potato acid phosphatase or calf intestine alkaline phosphatase which can successfully dephosphorylate a wide variety of phosphoproteins modified on several different amino acids. It should be noted that acid phos-phatases from animal tissues (e.g., bone, prostate, liver, and heart) represent a class of low-molecular-weight (18,000- to 25,000-kDa) protein tyrosine phosphatases that selectively dephosphorylate phosphotyrosine residues. These enzymes were defined as acid phos-phatases because they hydrolyzed p-ni-trophenyl phosphate (PNPP) at a pH optimum between 4.0 and 5.0. However, at physiological pH, these enzymes appear to specifically de-phosphorylate proteins and peptides containing phosphotyrosine residues. The mammalian acid phosphatases show no significant activity against phosphoserine/phosphothreonine-con-taining proteins.

With phosphoprotein substrates, potato acid phosphatase shows a preference for dephos-phorylating phosphotyrosine residues, followed by phosphothreonine residues. By comparison, this enzyme shows very low activity against phosphoserine-containing substrates. Wheat germ acid phosphatase can dephosphory-late phosphoserines but still shows significantly higher activity against phosphotyrosine and phosphothreonine (Van Etten and Waymack, 1991). In this regard, alkaline phosphatase is often preferred over potato or wheat germ acid phosphatase for dephosphorylation of phos-phoserine, the predominant protein modification in eukaryotic cells.

When a general phosphatase, such as alkaline phosphatase, is used, effects independent of protein dephosphorylation must be considered. For instance, alkaline phosphatase hydrolyses ATP and inhibits ATP-dependent processes, such as protein kinases and ATP-depend-ent ion channels that are also inhibited by protein dephosphorylation (Berger et al., 1993). To distinguish between these differing effects, use of immobilized alkaline phos-phatase is recommended. Agarose or dextran beads covalently linked to alkaline phosphatase should be washed extensively with the assay buffer to remove storage solutions and any unbound alkaline phosphatase. Following incubation with the substrate protein, immobilized alkaline phosphatase can be removed by centrifugation or filtration. Functional analysis of the substrate can then be undertaken without concern for the potential effects of the phos-phatase on such analysis. However, if the substrate is present in a particulate fraction (e.g., in subcellular organelles), it cannot be easily separated from the beads by centrifugation or filtration. In that case, the phosphatase should be inhibited with a general inhibitor such as sodium pyrophosphate. In contrast to the general phosphatases, protein phosphatases are inefficient at hydrolyzing ATP and do not hinder analysis of ATP-dependent processes.

Once phosphorylation of a substrate is established using a general phosphatase, the next step is to use protein phosphatases that have much higher activities against phosphoprotein substrates than either acid or alkaline phos-phatase and are highly selective for specific phosphoamino acids. Protein phosphatases 1, 2A, and 2B (PP1, PP2A, and PP2B) represent three major classes of protein serine/threonine phosphatases in eukaryotic cells. These enzymes differ in their subunit structure, substrate specificity, and regulation by divalent cations. PP1 and PP2A account for >90% of the total protein serine/threonine phosphatase activity in many cell extracts (Shenolikar and Nairn, 1991) and demonstrate a broad in vitro substrate specificity. Therefore, PP1 and PP2A are the most commonly used reagents for in vitro dephosphorylation of phosphoserine- and phosphothreonine-containing proteins. PP1 and PP2A are inhibited by microcystin-LR as well as several other recently discovered inhibitors, including okadaic acid, calyculin A, and tautomycin, albeit with differing IC50 values.

Inhibitors, such as okadaic acid (with IC50 for PP2A of 0.1 to 1.0 nM and for PP1 of 10 to 100 nM), calyculin A (with an IC50 for PP1 of 1 nM and for PP2A of 10 nM), and microcys-tin-LR (with an IC50 for both PP1 and PP2A of 0.1 nM) are used to exclude nonspecific effects of PP1 and PP2A. These inhibitors represent important experimental tools for establishing the physiological importance of reversible protein phosphorylation.

PP2B, also called calcineurin, is a Ca2+/cal-modulin-dependent phosphatase and demonstrates a narrow in vitro substrate specificity compared to PP1 or PP2A. The enzyme purified from mammalian tissues consists of a catalytic subunit that is tightly associated with a calcium-binding regulatory subunit. PP2B has no activity in the absence of 1 |M calmodulin and 1 mM CaCl2 and can be activated by either Ca2+/calmodulin or 1 mM MnCl2. Many highly purified PP2B preparations show no activity in the presence of Ca2+/calmodulin but are fully activated by Mn2+. The immunosuppressive drugs cyclosporin A and FK506 selectively inhibit PP2B in vivo and in vitro.

PP2B in the presence of Ni2+ ions and PP2A in association with an endogenous regulator protein or viral antigens can dephosphorylate proteins and peptides containing phosphoty-rosines. However, under the conditions described in Basic Protocol 2, PP1, PP2A, and PP2B are highly selective for dephosphoryla-tion of phosphoserine and phosphothreonine residues. PP1 and PP2A represent the major phosphohistidine phosphatase activity measured in tissue extracts, but the regulatory importance of this modification has not yet been established.

Critical Parameters

Some commercial preparations of potato acid phosphatase contain contaminating protease activity. Because phosphorylation sites reside near the surface of the substrate protein, they are often accessible to proteases, so functional changes that result from limited prote-olysis can be incorrectly attributed to dephos-phorylation. To limit the effects of proteases, high enzyme concentrations or prolonged incubations with the general phosphatases should be avoided. Aside from including protease inhibitors in the reaction, two strategies should be considered to distinguish proteolysis from dephosphorylation. Formation of a complex between trichloroacetic acid-soluble [32P]phosphate and ammonium molybdate (Support Protocol) is particularly useful in distinguishing [32P]phosphate from 32P-labeled phosphopeptides. The use of phosphatase inhibitors in control reactions also identifies those effects that can be attributed to reactions in which there are contaminating proteolytic enzymes.

Commercial preparations of PP2A and PP1 contain only the free catalytic subunits. In vivo these subunits associate with regulatory subunits, which modulate their substrate specificity. However, the catalytic subunits are the most readily purified to homogeneity and can be stored for prolonged periods without significant changes in their enzymatic properties. Both PP1 and PP2A catalytic subunits retain high activity in the absence of divalent cations; PP2A activity may be further stimulated by 1

Post-Translational Modification: Phosphorylation and Phosphatases

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