Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth.

cyclic peptides nodularin (71), microcystin LR (120, 178), and motuporin (57). Okadaic acid, tautomycin, and calyculin A are complex organic molecules that are membrane permeant, allowing their use in intact cells to inhibit PP1 and PP2A. Other major advances have been the application of recombinant DNA technology and yeast genetics to identify the contribution of these enzymes to cell physiology. Lists of the potential functions of protein serine/threonine phosphatases in cellular regulation, particularly in such aspects as carbohydrate metabolism and muscle contraction, have been described in recent reviews (21, 35, 235). The following discussion focuses on recent examples of how these enzymes function in the I. INTRODUCTION

Biochemical signal transduction pathways transmit information arriving at the plasma membrane to the interior of the cell. Signal transduction pathways are also responsible for coordinating cellular activity during complex functions. Alterations in the signals generated by these pathways can cause profound cellular changes such as those leading to differentiation or cellular transformation. Reversible covalent modification of proteins by phosphorylation and dephosphorylation plays a dominant role in controlling the activities of proteins involved in signaling (156, 277). The net activity of a phosphoprotein at any given time depends on the proportion of molecules in the phosphorylated and dephosphorylated states, which in turn depends on the relative activities of protein kinases and protein phosphatases. Although it has been clear for many years that the activities of protein phosphatases are crucial components of cellular signal transduction pathways, very little information has been available on how individual enzymes were involved in regulation of cell function. The recent application of molecular genetic methods and the development of biochemical assays for complex cell functions have provided exciting new insights into the roles of these enzymes. This review describes recent developments in the understanding of the physiological roles of protein serine/threonine phosphatases by focusing on emerging themes regarding the regulation and functions of this enzyme family.

II. PROTEIN SERINE/THREONINE PHOSPHATASES COMPRISE A GENE FAMILY

Two major families of protein phosphatases are present in eukaryotic cells: protein serine/threonine and protein tyrosine phosphatases. The tyrosine phosphatases are a family of intracellular and receptor-like enzymes that play important roles in signaling pathways involving tyrosine kinase-linked oncogenes and growth factor receptors. Despite their similar enzymatic functions, the tyrosine phosphatases have no primary sequence similarity to the protein serine/threonine phosphatases. In general, members of these families are specific for dephosphorylation of serine/threonine or tyrosine residues. However, at least one protein serine/threonine phosphatase (type 2A) has a low level of tyrosine phosphatase activity (31, 90), and a vaccinia virus-encoded protein tyrosine phosphatase dephosphorylates both phosphotyrosine and phosphoserine (97). The protein tyrosine phosphatases have been the subject of several recent reviews (27, 76, 127, 226, 255-257).

The number of distinct forms of protein serine/threonine phosphatases that have been identified is considerably smaller than the number of protein serine/threonine kinases. The most recent estimates indicate that at least 200 protein kinases have been identified (104) and that there may be as many as 1,000 protein kinase genes in eukaryotes (126). As of 1990, a total of 15 clearly distinct but related protein serine/threonine phosphatase catalytic subunit genes had been identified in eukaryotes and bacteria (37). A distinct family of protein serine/threonine phosphatase |protein phosphatase type 2C (PP2C)~ has also been identified (250) that contains at least two members (185). While the numbers of characterized phosphatase genes will undoubtedly increase, one must conclude that a single phosphatase catalyzes the dephosphorylation of proteins phosphorylated by more than one protein kinase. Studies of protein phosphatases and their catalytic subunits in vitro have confirmed this. Despite the limited numbers of phosphatase genes currently known, the multiplicity of enzyme forms is increased significantly by the association with multiple types of regulatory subunits.

A. Classification of Protein Phosphatases

The classification of the protein serine/threonine phosphatases is a confusing issue, and there is not yet a general consensus on the best system. The first classification scheme, and the one that is used most widely, is based on biochemical differences in the activity and regulation of individual enzymes (130, 131). The criteria for grouping individual enzymes into classes include the relative activity toward the |Alpha~- and |Beta~-subunits of phosphorylase kinase and sensitivity to two of the inhibitor proteins, inhibitor 1 and inhibitor 2. Type 1 protein phosphatases (PP1) preferentially dephosphorylate the |Beta~-subunit of phosphorylase kinase and are sensitive to both inhibitor 1 and inhibitor 2. The type 2 enzymes preferentially dephosphorylate the |Alpha~-subunit of phosphorylase kinase and are insensitive to either of the inhibitor proteins. The type 2 enzymes are further divided on the basis of structural and regulatory properties. Type 2A phosphatase (PP2A) represents a class of oligomeric enzymes with no obvious requirements for ions or cofactors. Type 2B phosphatases (PP2B), also known as calcineurin, are regulated by |Ca.sup.2+~/calmodulin. A fourth class of protein serine/threonine phosphatase, type 2C, requires |Mg.sup.2+~ for activity. The cloning of individual forms of protein serine/threonine phosphatases has confirmed this functional classification. In general, members of the functionally defined classes have primary sequences that are much more closely related to each other than to enzymes of a different class. Cloning and sequencing of catalytic subunit cDNAs have also shown that each class has at least two isozymes and that three of them (PP1, PP2A, and PP2B) are structurally related. Although this classification is a useful one, it is clearly inadequate, since a number of new phosphatase genes have been identified that do not easily fit into one of the known classes.

An effort has been made to simplify the classification of protein serine/threonine phosphatases and provide a rational basis for naming newly discovered enzymes. A numerical nomenclature system for the common names of enzymes was proposed at the Federation of American Societies for Experimental Biology Summer Conference on Protein Phosphatases (July 19-24, 1992). With this system, protein phosphatase 1 would be PP1, protein phosphatase 2A would be PP2, and protein phosphatase 2B would be PP3. Because protein phosphatase 2C is in a distinct gene family, it would be designated as MP1. The individual subunits and isoforms of each class would be designated by a letter followed by a lower case greek letter (e.g., the |Alpha~-form of the catalytic subunit of protein phosphatase 1 would be designated PP1C|Alpha~). We have utilized this new nomenclature system in several places in this review, most notably in Tables 2 and 4, in a effort to introduce it. However, most of the text relies on the traditional names to avoid confusion.

B. Structures and Isozyme Diversity

1. Protein phosphatase 1

Type 1 protein phosphatases consist of multimeric structures composed of a catalytic subunit complexed to a number of accessory subunits. The free catalytic subunit has not been detected in cell or tissue extracts, suggesting that little, if any, uncomplexed catalytic subunit is present in vivo. Formation of heteromeric complexes plays an important role in regulating the activity of the catalytic subunit. The oligomeric forms and subunit compositions of PP1 are summarized in Table 1. The first native form of PP1 to be characterized was isolated as a MgATP-dependent protein phosphatase composed of a complex between the PP1 catalytic subunit and inhibitor 2 (15, 141,261). This form of PP1 has low intrinsic activity and is activated subsequent to phosphorylation of the inhibitor 2 component by glycogen synthase kinase 3. This form of PP1 has also been referred to as |PP1.sub.I~ and as the MgATP-dependent protein phosphatase and is primarily a cytosolic form (35).

The PP1 associated with glycogen (|PP1.sub.G~) consists of a stoichiometric complex between the PP1 catalytic subunit and a glycogen binding protein termed the G subunit (245). As discussed in section IIIA2, the G subunit targets the PP1 catalytic subunit to glycogen and regulates its activity. Type 1 protein phosphatase activity associated with the sarcoplasmic reticulum in skeletal and cardiac muscle is similar, if not identical, to |PP1.sub.G~(124, 177), suggesting the G subunit (also termed the |R.sub.Gl~ subunit) also plays a role in binding the catalytic subunit to membranes. A dual function for the G subunit (association with glycogen and membranes) is supported by the predicted amino acid sequence, which contains a very hydrophobic region near the COOH-terminus that could serve as a membrane spanning or anchoring domain (252). The GAC1 gene of Saccharomyces cerevisiae has recently been shown to be homologous to the G subunit (80). Elevated levels of GAC1 protein cause hyperaccumulation of glycogen, while disruption of the gene causes reduced glycogen levels, indicating that the G subunit and its yeast homologue play direct roles in glycogen metabolism.

The PP1 catalytic subunit is also associated with the actomyosin contractile apparatus in various types of muscle. The association of a protein phosphatase capable of dephosphorylating the regulatory light chain subunit (P-light chain)…