1002 protein phosphatases?

Further

ANNUAL REVIEWS

Quick links to online content Annu. Rev.Cell Bioi.

1992. 8:463-93 Copyright © 1992 by Annual Reviews Inc. All rights reserved

1002 PROTEIN

Annu. Rev. Cell. Biol. 1992.8:463-493. Downloaded from www.annualreviews.org Access provided by McGill University on 02/07/15. For personal use only.

PHOSPHATASES? Harry Charbonneau Department of Biochemistry, Purdue U niversity, West Lafayette, Indiana 47907

Nicholas K. Tonks Cold S pring Harbor Laboratory, Demerec Building, Cold S pring Harbor, N ew York 1 1 724 KEY WORDS: protein tyrosine phosphatases, tyrosine phosphorylation, signal transduction, dephosphorylation. cell cycle, transformation

CONTENTS INTRODUCTION

THE PTP GENE FAMILY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

463 464

STRUCTURE AND FUNCTION OF PTP DOMAINS . . . . . . . . . . . . .. . . . . . . . . . .

473

REGULATION OF PTP ACTIVITY . . . . Ligands for Receptor-like PTPs . . . . . . Phosphorylation of PTPs . .... . . . .. Role of Targeting Domains in PTPs . .

474 474 475 476

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PHYSIOLOGICAL FUNCTION OF PTPS . . . . . . . The Role of CD45 in Lymphocyte Activation . . . Potential Role of PTPs in Cell Adhesion . .... . PTPs and the Control of the Timing of Mitosis . The Role of PTPs in Transformation . . . . . . . . . CONCLUSION

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477 477 480 482 486 487

INTRODUCTION The concept that protein function can be modulated by reversible phosphor ylation is now well established and appears to impinge on essentially all aspects of cell physiology (Fischer & Krebs 1 989). The observation that the receptors for a variety of hormones and growth factors, as well as the transforming proteins of certain acutely oncogenic retroviruses, possess intrinsic kinase activity with specificity for tyrosyl residues suggests important

463 0743-4634/9211115-0463$02.00


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CHARBO N N EAU & TO N KS

new roles for protein phosphorylation in the control of cell growth, prolifer ation, and differentiation. Although the protein kinases have provided a focus of substantial research effort, one should not ignore the fact that the net level of phosphate detected in a target substrate in vivo reflects the competition between the action of protein kinases and phosphatases . Thus a complete understanding of the physiological role of tyrosine phosphorylation and its potential as a mechanism for the reversible modulation of protein function must necessarily encompass the characterization of the protein tyrosine phosphatases (PTPs) in addition to the protein tyrosine kinases (PTKs). Initial studies of tyrosine dephosphorylation indicate that the type II ser/thr phospha tases, but not typc I, possess significant intrinsic PTPase activity. Whether they function as PTPs in vivo, where phosphoseryl and phosphothreonyl residues are present in vast excess over phosphotyrosine, remains unclear. Similarly acid and alkaline phosphatases also dephosphorylate tyrosyl residues in vitro, but it is not known whether they can be classified as PTPs in vivo. This review focuses on a novel class of phosphatases that are highly specific for tyrosyl residues and dephosphorylate phosphotyrosyl proteins in vivo. The structural features of members of the family and possible modes of regulation are described. In addition ,several important aspects of cell biology in which a role for PTPs has either been implicated or verified are discussed.

THE PTP GENE FAMILY The protein tyrosine phosphatases constitute a rapidly growing and diverse family of enzymes that exist as both transmembrane, receptor-like and nonreceptor forms. Despite exceptional diversity in size and structural organization, a common evolutionary origin for this protein family is demonstrated by the presence within each isoform of at least one conserved segment of 240 residues. A common and defining feature of the PTP family is this homologous sequence, which is assumed to delineate an independently folding, catalytic domain. As described below, there is now considerable evidence to support this contention. Most PTPs are multidomain proteins (Figure 1) that contain at least one catalytic domain as well as additional unrelated sequences of variable size. The combination of catalytic domains with a wide variety of structural motifs accounts for the diversity among the PTPs . Within nonreccptor isoforms, noncatalytic sequences confer distinct modes of regulation and target the PTPs to specific subcellular compartments. Presumably the fusion of PTP domains with a transmembrane segment and a variety of extracellular structural motifs has created receptor-like isoforms that each have the ability to respond to distinct ligands. The degree to which diversity among PTP domains may reflect differences in substrate recognition is unclear since little is known about

1002 PROTEIN PHOSPHATASES?

465


substrate specificity. Similar chimeric or mosaic structures characterize the protein kinase family. Mosaic proteins are thought to have been constructed through a process involving exon shuffling (Patthy 1991). The first PTP primary structure was determined by protein sequence analysis. Since then, 25 additional PTPs (excluding species homologues) have been identified with 10 of these being transmembrane isoforms (Tables 1 and 2). Although several were discovered as pre-existing sequences in databases, most were isolated as cDNA clones using strategies employing peR or low

HPTP�

LAR

DPTP HPTPj.l. C045 DPTP 99A HPTPo:

Extracellular

Intracellular

�

Type I

Type II

Type III

Type IV

I I r� I I �� iIi i'.

'�w .

PTP1 B

Figure 1

PTP H1

.�

STEP

YOP 2b

cdc 25A

P TP'PEST

The conserved catalytic domains are shown in black. The PTPs can be categorized

as transmembrane. receptor-like

or

nontransmembrane molecules . At the present time the

receptor-like species can be subdivided into four types based on the structure of their extracellular segments. Type I represents the

CD4S family. multiple isoforms of which arise from differential

splicing of a primary mRNA transcript of a single gene; three exons encoding sequences at the extreme N-terminus (horizontal lines) are differentially expressed. Type II contain immun oglobulin-like (diagonal lines) and tandem fibronectin type III-like repeat domains (stippled); this category includes LAR (leukocyte common antigen-related), DLAR, DPTP. and HPTPf-L.

Type III bear multiple fibronectin type III-like repeats. Some type III isoforms such as HPTPI3 have only one internal PTP domain. Type IV isoforms such as Ol and E have small glycosolated extracellular segments. Multiple nontransmembrane forms have also been identified. Many of the nonreceptor PTPs bear noncatalytic segments that are structurally related to other well characterized proteins. The position and relative size of these noncatalytic domains are shown as boxes containing distinct symbols; noncatalytic regions that have similar sequences are designated with identical patterns. The noncatalytic segments that have been identified include two SH2 domains in PTP IC, and band 4. 1 homology domains in PTPH 1 , an apparent lipid-binding domain in MEG2, and segments containing PEST sequences in PEP and PTP-PEST. In TC-PTP and PTPlB, the C-terminal noncatalytic segments appears to play a role in modulating activity and controlling subcellular localization. PTPI, STEP, and Yop2b have noncatalytic sequences that are apparently unrelated to sequences in the databases. The protein from vaccinia virus, V H I , is much smaller than the other PTPs and presumably encodes only essential sequences within the

catalytic domain. A number of cDNAs encoding additional PTPs have been identified. but are not included. Diagrams are based on sequences presented in the references listed in Tables 1 and 2.


466

CHARBON NEAU & TON KS

stringency hybridization. The majority of isoforms are derived from human or rodent sources, but the presence of PTP genes in insects, a protochordate Styela pUcata (Matthews et al 1991), C. elegans (Matthews ct a1 1991; A. J . Flint e t aI, unpublished observations), and yeast (Ottilie e t a l 1 99 1 ; Guan et al 1991b; Ota & Varshavsky 1992) indicate that these enzymes are probably ubiquitous among eukaryotes. The discovery of receptor-like PTPs followed the sequence determination of PTPIB, a nonreceptor isoform purified from human placenta (Tonks et al 1988b). The PTPIB sequence was homologous to two tandem domains within the cytoplasmic tail of the leukocyte common antigen, CD45 (Charbonneau et al 1988). Subsequently, CD45 was demon strated to have intrinsic tyrosine phosphatase activity (Tonks et al 1988a). CD45 has receptor-like properties since it has a large, heavily glycosylated external domain with the potential to bind surface ligands on either the same or adjacent cells (Thomas 1989). The structural features of CD45 suggest that it may function as a receptor-linked PTP that could transduce external signals in a manner similar to the receptor tyrosine kinases (Charbonneau et al 1988). Thus CD45 and the other recently identified transmembrane PTPs may represent a new class of signal transduction molecules that activate intracellular signaling cascades by dephosphorylation of protein tyrosyl residues. The distinguishing features of the receptor-like PTPs are the presence of a single transmembrane segment and two tandem PTP domains within the cytoplasmic tail (Figure 1; Table 1). Only HPTPI3 and DPTPIOD have single PTP domains. In contrast to the similarity within the internal domains, there is considerable diversity among extracellular segments of receptor-like PTPs. Fischer et al (1991) suggested that these PTPs are best distinguished on the basis of their external segments. For the purposes of discussion, we utilize this classification scheme (Figure 1). The type I transmembrane PTP, CD45, has a large extracellular segment that appears to be unrelated to other protein sequences. The external domain has an N-terminal segment containing O-linked carbohydrates; variations in the length of this segment arise from alternative splicing and result in changes in the extent of glycosylation. A large cysteine-rich region (360 residues) containing N-linked carbohydrates is juxtaposed to the transmembrane segment and has the hallmarks of a ligand-binding motif (Thomas 1989). The type II isoforms contain one to three tandem Ig-like domains near their N-termini that are adjacent to two to ten tandem fibronectin type III-like repeats (FNIII). These structural features resemble the N-CAM family (e.g. N-CAM, NG-CAM, contactin, fasciclin II, etc) of cell adhesion molecules, thus implying that these PTPs may have similar ligand-binding properties and may be involved in cell adhesion phenomena. The type II PTP, LAR, undergoes posttranslational processing so that it is expressed as a complex of two non-covalently associated subunits


Table 1

Receptor-like PTP isofonns

PTP'

Organism

Characteristic structural and functional features

CD45

Human

Restricted to hematopoietic cells Essential com-

Type

Thomas et al 1989; Charbonneau et al

_

Rat

ponent in T- and B-cell activation_ Variants

Mouse

arise from alternative splicing of N-terminal

References

1989; Tonks et al 1988a

segment. A large cysteine-rich region is juxtaposed to the transmembrane segment. LAR

Human

3 Ig-like repeats; 9 FN III-like repeats

II

Streuli et al 1988; Pot et al 1991

Rat DLAR

Drosophila

3 Ig-Iike repeats; 10 FN III-like repeats

II

Streuli et al 1989

DPTP

Drosophila

2 Ig-like repeats; 2 FN III-like repeats

II

Streuli et al 1989

;;

PTP�

Human

1 Ig-1ike repeats; 4 FN III-like repeats

II

Gebbink et al 1991

'"0 ::0 0 >-3

16 FN III-like repeats; one PTP domain

III

Krueger et al 1990

Mouse HPTP{:l

Human

0 h)

� Z

'"0

DPTP99A

Drosophila

2 FN III-like repeats

III

Yang et al 1991; Tian et al 1991;

Hariharan

et

a\ 199\

DPTPIOD

Drosophila

12 FN III-like repeats; one PTP domain

III

Yang et al 1991; Tian et al 1991

RPfP a

Human

Small external segment (123 residues)

IV

Sap et al 1990; Krueger et al 1990; Kaplan et al 1990; Jirik et al 1990;

Mouse

:I:: 0

'-"'"0

:I:: >

�

en tIl en

-...:l

Matthews et al 1990 HPTP"

Human

Small external segment (27 residues)

'Obvious species homologues are grouped together_

IV

Krueger et al 1990

.j:>. C\ ---.J


468

CHARBONNEAU & TONKS

(Streuli et al 1 992). A 1 50-kd subunit (E) is derived from the N-CAM-like external segment; the smaller 85-kd subunit (P) includes the two PTP domains, transmembrane region, and a short external segment that interacts with the large subunit. The E-subunit is shed from the cell surface during growth in a density-dependent fashion, but the regulatory consequences for the catalytic P-subunit remain to be established. The type III receptor-like PTPs are distinguished by having only tandem FNIII-like repeats. The type IV group includes HPTPe and HPTPa, which are characterized by having short extracellular segments (27 and 123 residues, respectively) that may be highly glycosylated (Daum et aI 1 991). Although these segments are somewhat short, a receptor function for type IV PTPs cannot be excluded until it is known whether the external domains are associated with additional subunits that might confer ligand-binding capabilities . Partial eDNA clones (Krueger et a1 1990; Kaplan et a1 1 990) have been isolated for three putative receptor-like isoforms (HPTP8, HPTP,,(, and HPTP�). HPTP8 is closely related to LAR in its internal PTP domains and in the partial extracellular sequence that contains tandem FNIII-like repeats. The external sequence of HPTP,,( and 8 are unknown . Unlike their receptor-like counterparts, nonreceptor PTPs have a single catalytic domain and unrelated, noncatalytic sequences of variable length that are positioned at either the N- or C-terminal ends of the molecule. Sequence similarities to other well characterized proteins and, in some cases, direct experimental evidence suggest that one of the major functions of noncatalytic regions is localization to subcellular compartments that may direct PTPs to specific sub sets of proteins containing phosphotyrosine. However, these segments may also be involved in regulating enzyme activity. In some cases, the nonreceptor PTPs may be divided into subfamilies on the basis of similarities among their noncatalytic domains. For example, PTPHI and MEG-Ol from humans (Table 2) both contain N-terminal segments that are homologous to the family of proteins that includes band 4.1, talin and ezrin (Figure 1 ). Given the sequence similarity in their N-terminal domains, it is not surprising that their catalytic domains are 65% identical. In erythrocytes, band 4. 1 promotes the association of actin and spectrin and through its N-terminal segment binds to the transmembrane protein glycophorin (Bennett 1 989). A similar model has been proposed for the interaction of talin and integrin. The presence of such a domain raises the possibility that these enzymes may be localized to junctions between actin stress fibers and the inner aspect of the plasma membrane where they may modulate cytoskeletal dynamics. The catalytic domains of the T-cell PTP and PTP1 B display a high degree of similarity (74% sequence identity). Although their C-terminal, noncatalytic sequences (100 residues) have retained significant structural similarity, they have diverged more rapidly than the catalytic domains . Both enzymes have a



469

hydrophobic segment that comprises the last 20 carboxy-terminal residues (Cool et al 1 989). Although the similarity is weak, the C-terminal sequences from these two PTPs indicate that they may have related functions . Thus far, this seems to be the case. As discussed in detail below, there is mounting evidence that the noncatalytic domains direct both enzymes to membranes. With the T-cell PTP, there are data to suggest a role for this segment in regulation of enzyme activity. A PTP referred to as PTPIC (Shen et al 1991), SH PTPI (Plutzky et al 1992), HCP (Yi et al 1992), and SHP (Matthews et al 1992) is characterized by the presence of two SH2 domains (src homology domain 2) at its N-terminus. SH2 domains bind to phosphotyrosyl residues in proteins and are present within the family of src-like kinases, as well as other proteins with roles in signaling pathways such as phospholipase C"" ras GAP (GTPase-ac tivating protein), v-crk, tensin, etc (for review , see Koch et aI 1991). Thus it is possible that tyrosine phosphorylation of certain proteins (e g in response to mitogens or growth factors) can trigger the translocation of PTPs containing SH2 domains to specific intracellular sites and thereby restrict the substrates that they can dephosphorylate . Another level of control may exist if activity is modulated by the binding of phosphotyrosyl residues to the SH2 domains of these PTPs, e . g. through an autoinhibition mechanism if the PTP is itself phosphorylated on tyrosyl residues. A PTPase termed MEG2 (Gu et al 1992) has been identified in which the catalytic domain is linked to an N-terminal segment displaying 28% identity to retinaldehyde-binding protein, and 24% identity to SEC14p, a phos phatidylinositol transfer protein of S. cerevisiae. This raises the intriguing possibility that the activity of this enzyme may be controlled by binding of a lipid moiety to the N-terminal domain. In addition, two distinct gene products, termed PEP (Matthews et a1 1992) and PTP-PEST (Q. Yang et aI, submitted), have been isolated that feature noncatalytic C-terminal segments that are rich in Pro, GIu/Asp, and Ser/Thr residues. Such PEST sequences characterize proteins with very short intracellular half-lives (Rogers et al 1986), which suggests that these noncatalytic domains may target the enzymes for rapid degradation in vivo. HePTP, STEP, and PYP1 from S. pombe and PTP2 from S. cerevisiae have N-terminal noncatalytic sequences for which there is no apparent similarity to other proteins and no known function. STEP is expressed primarily in rat brain and is most enriched in the striatum, whereas expression of HePTP is restricted to the thymus and spleen. The PTP domain of PTPl from S. cerevisiae encompasses most of the molecule and no additional functional domains are apparent. Upon searching the sequence databases, Guan & Dixon (1990) noted homology between the PTPs and YOP2b, a protein encoded by a plasmid .

.


Table 2

ti

o

Nonreceptor PTP isoforms Characteristic structural and functional

PTpa

Organism

features

References

PTP IB

Human

C-terminal regulatory domain with 20 resi

PTP I

Rat

Charbonneau et al 1989; Guan et al 1 990;

due hydrophobic tail

Chernoff et al 1 990; Brown-Shimer et al 1 990

TC-PTP

Human Mouse

C-terminal regulatory domain with 20 resi due hydrophobic tail

Cool et al 1989; Swarup et al 1 99 1 ; Mosinger et a l 1992

Rat PTP HI

Human

Band 4. I-like N-terminal domain

Yang & Tonks 1 99 1

MEG-OI

Human

Band 4. I-like N-terminal domain

G u e t al 1991

PTP IC

Human

Two N-terminal SH2 domains

Shen et al 1991; Yi et al 1992; Plutzky

HCP

Mouse

et al 1992; Matthews et al 1 992

SH PTPI SHP HePTP

Human

N-terminal noncatalytic segment of

Zanke et al 1 992

unknown function STEP

Rat


Lombroso et al 1991

unknown function MEG-2

Human

Retinaldehyde-binding protein-like N-terminal domain

Gu et al 1 992

g � o:l

o Z Z tTl ;l> c:: P."

d z :;>::

�


PEP

Mouse

C-terminal domain of

=

500 residues

Matthews et al 1992

containing PEST sequences PTP-PEST

Human

C-terminal domain of

=

1 90 residues

Q. Yang et ai, submitted

containing PEST sequences pypl+

S.

pombe


Ottilie et al 1 991

unknown function PTP 1

S. cerevisiae

None

Guan et al 1 9 9 1 b

PTP 2

S. cerevisiae

N-terminal noncatalytic segment of un

Ota & Varshavsky 1992

known function Yop 2b/5 1

Yersinia

Required for pathogenesis of bacteria of this genus

Guan & Dixon 1990

Distantly related to other PTPs smaller

Guan et al 1 99 1 a

than typical PTP domain cdc 25

Drosophila

Distant relative of other PTPs

cdc 25 A, B,C

Human

Dephosphorylates regulatory Tyr residue of cdc2

a

Obvious species homologues are listed together.

;g

Z

unknown function Vaccinia virus

N

�


VH I

§

�

o til

Gautier e t a l 1 99 1 ; Dunphy & Kumagai

1 9 9 1 ; Strausfeld et al 1 99 1 ; Galaktionov

& Beach 1991

�

�

til tIl til . ..,

.j:>. -...]


472

CHARBONNEAU & TONKS

required for the virulence of the bacterium, Yersenia pseudotuberculosis. Bacteria of the genus Yersenia are pathogenic to humans and rodents; Y. pestis is responsible for the plague or black death. Expression of the YOPSI gene product from Y. enterocolitica demonstrated that these genes encode active PTPs (Guan & Dixon 1990). YOP2b is required for pathogenecity of Y. pseudotuberculosis and causes the dephosphorylation of protein tyrosyl residues in host cells (Bliska et al 1991). The Yersenia PTPs also contain noncatalytic N-terminal sequences of unknDwn function. A database search also revealed that the VH1 gene of vaccinia virus encodes a 20-kd protein that is distantly related to the PTPs (Guan et al 1991a). Expression of the VHl gene produces an enzyme that not only dephos phorylates phosphotyrosine, but also phosphoserine residues in artificial substrates. It is not known whether VH1 is involved in viral replication or pathogenesis. Recently, several groups have shown that the Drosophila and human cdc25 genes encode tyrosine phosphatases that specifically dephos cdc2 phorylate p34 , a protein kinase that must be activated for initiation of mitosis (Gautier et a11991; Dunphy & Kumagai 1991; Strausfeld et aI1991). The activity of human cdc25 appears to be positively regulated by B-type cyclins (Galaktionov & Beach 1991). The role of protein tyrosine dephos phorylation in cell cycle control is discussed in greater detail below. Both VHl and cdc25 display relatively low sequence similarity to other PTP domains. The complete VHl protein has 170 residues and is considerably smaller than the other domains. The greatest similarity between VH1, cdc25, and the other domains is restricted to about 30 residues surrounding the consensus HCXAGXXR motif. It seems plausible that cdc25 may represent a case of convergent evolution; however, using the profile method of analysis, Gautier et al (1991) argue that the similarity between cdc25 and the other PTPs is more extensive and that there is a significant evolutionary relationship. If VHl and cdc25 share common ancestry with the other PTPs, it is clear that they must have diverged long ago and/or at a very rapid rate. Alternative mRNA splicing also contributes to the diversity of PTP isoforms, Differential usage of three exons encoding sequences at the N-terminus of CD45 gives rise to six isoforms (Thomas 1989). Evidence of alternative splicing has been reported for three other receptor-like isoforms, RPTPa DPTPlOD, and DPTP99A (Matthews et al 1990; Kaplan et al 1990; Yang et al 1991). It now appears that an alternately spliced variant of the T-cell PTP does not have the last 28 C-terminal residues that include the distinctive hydrophobic region (Mosinger et al 1992), thus raising the possibility that this variant might have a distinct intracellular location. Surprisingly, the PTP gene family is not structurally related to catalytic subunits from either the type lItype 2A or type 2C protein Ser/Thr phosphatase families (Cohen 1989). Furthermore, sequences for several alkaline and acid



473

phosphatases of broad specificity bear no resemblance to those of the PTPs. Thus there are at least three different protein phosphatase gene families that appear to have distinct evolutionary origins. Interestingly, both Ser/Thr-spe cific and Tyr-specific protein kinases share common progenitors. Why at least three distinct gene families evolved for the removal of phosphate from proteins remains an intriguing question. At present, the number of distinct PTP genes appears to be much greater than the number of protein Ser/Thr phosphatases. In fact, there is every indication that the number of distinct PTPs may approach that for the PTKs. This might reflect fundamental differences in the mecha nisms controlling protein tyrosine and protein serine/threonine dephosphoryla tion reactions.

STRUCTURE AND FUNCTION OF PTP DOMAINS The segment conserved among all PTPs has been shown to contain the active site of these enzymes. Multiple sequence alignments indicate that there are about six highly conserved regions within the PTP domain. Of particular interest is a segment of 11 residues located near the carboxy-terminus of the domain with the consensus sequence (I/v)HCXAGXXR(S/T)G. The cysteine within this motif is found in all PTPs except the C-terminal domains of the receptor-like isoforms, HPTP), and DPTP99A, where it is replaced by aspartate. Several studies employing site-directed mutagenesis to replace this cysteine have demonstrated that it is required for activity (Streuli et al 1989, 1990; Guan & Dixon 1990; Guan et al 1990, 1991b; Bliska et al 1991; Pot et al 1991). Pot & Dixon (1992) have shown that inactivation of the cytoplasmic domain of rat LAR by iodoacetate is correlated with the near stoichiometric labeling of the protein. The residue labeled by radioactive iodoacetate was shown to be the cysteine residue of the conserved motif. Furthermore, Guan & Dixon (1991) have isolated a phosphoenzyme intermediate with rat PTPlB that is absent in mutants in which the essential cysteine residue is altered. These data indicate that the conserved cysteine is located at the active site of the PTPs and participates in the dephosphorylation reaction by serving as a nucleophile. The functional significance of the two-domain arrangement that character izes the receptor-like PTPs is not yet understood and appears to be somewhat controversial. This unusual topology raises several questions. Do both domains have intrinsic PTP activity? What role does each domain play in mediating the cellular function of these PTPs? Several groups have used site-directed mutagenesis to probe the catalytic properties of each of the two domains using artificial substrates such as myelin basic protein, several short peptides (e.g. RR-src, angiotensin II), and p-nitrophenyl phosphate. With these non-


474

CHARBONNEAU & TONKS

physiological substrates, it is impossible to know whether failure to detect activity results from an intrinsically inactive domain, or from use of substrates that are not recognized or hydrolyzed at detectable rates. Streuli et al (1989, 1990) found that replacement of the conserved cysteine in the HCXXG motif of domain II in human LAR and CD45 had little or no effect on PTP activity relative to wild-type, whereas no activity was detected when the cysteine of domain I was replaced. Expression of constructs encoding only domain I of LAR were nearly fully active, whereas constructs for domain II alone showed no measurable activity (Streuli 1990). These results suggest that domain II cannot dephosphorylate these substrates. By replacing the critical cysteine residue in each of the two domains in rat LAR, Pot et al (1991) obtained similar results; however, they concluded that a small but significant activity (about 1% of wild-type) remained following replacement of the cysteine of domain I, which leaves open the possibility that domain II could use the substrates tested. By expressing each domain alone, Wang & Pallen (1991) showed that domain II of HPTPa could hydrolyze p-nitrophenyl phosphate and dephosphorylate a synthetic peptide, RR-src, albeit at a very low rate relative to a double domain construct. These data suggest that domain II of HPTPa may have a functional active site with a high degree of substrate specificity. A mutational analysis of CD45 by Johnson et al (1992) indicated that both PTP domains and the membrane proximal segment were required for activity. Both Streuli et al (1990) and Wang & Pallen (1991) obtained evidence consistent with interactions between the two domains that might be indicative of an important means of regulation, in that mutations in domain II appeared to alter the specificity of domain l.

REGULATION OF PTP ACTIVITY The members of the PTP family share a high specific activity (one to three orders of magnitude in excess of that of the PTKs) (Tonks et a11988c, 1990), a high affinity for substrate and, with the exception of VHl and cdc25 , appear to be specific for tyrosyl residues. This suggests that these enzymes have the potential to represent a formidable barrier to the action of PTKs in vivo. In view of this one might anticipate tight control of PTP activity.

Ligands for Receptor-like PTPs For the receptor-like isoforms there is obviously the potential for modulation of activity by ligand binding. However, it should be emphasized that the direct evidence supporting the presumed receptor function of transmembrane PTPs is scant. The difficulty in identifying potential ligands concerns the anticipated effect on phosphatase activity. Unlike PTKs such as the EGF receptor, solubilized and purified receptor-like PTPs display considerable activity


1002 PROTEI N PHOSPHATASES?

475

against artificial substrates, apparently in the absence of ligand, which suggests that receptor-like isoforms may be constitutively active in vivo (Tonks et al 1990; Daum et al 1991). Rather than activating the enzyme, perhaps ligand binding inhibits activity or even modulates enzyme function by controlling its localization in the membrane, thus restricting interactions with substrates or other regulatory factors. A potential ligand for the 180-kd CD45 isoform (CD45RO) has recently been identified as CD22, a cell-surface antigen of B-cells (Stamenkovic et al 1991). The physiological significance of the CD22-CD45 interaction is not yet clear, but it may provide a mechanism for communication between B and T-cells (Stamenkovic et al 1991). Thus far nothing is known about the effects of CD22 binding on either PTP activity or the association of CD45 with other membrane proteins. The specificity of CD22 for one particular CD45 isoform also indicates that other ligands remain to be identified.

Phosphorylation of PTPs Phosphorylation provides an additional mechanism for regulation. Most available data refer to CD45, which is known to be phosphorylated in vivo on both serine (Omary & Trowbridge 1980) and tyrosine (Stover et aI1991). Protein kinase C (PKC) appears to be one kinase responsible for serine phosphorylation since CD45 is a substrate in vitro and is phosphorylated in vivo in response to treatment with phorbol ester (Autero & Gahmberg 1987). Yamada et al (1990) reported a decrease in the PTP activity of CD45 isolated from TPA-treated human peripheral blood lymphocytes, but this was not corroborated by Ostergaard & Trowbridge (1991). The enzyme is also phosphorylated in vitro by casein kinase 2 and glycogen synthase kinase 3 , but no effect on activity was detected (Tonks et al 1990). Ostergaard & Trowbridge (1991) have observed that ionomycin-induced increases in the 2+ levels of intracellular Ca of T-cells results in a decrease in CD45 PTP activity that coincides with a loss of phosphoserine. These results suggest that ci+ -dependent dephosphorylation regulates activity in vivo, but it is not yet 2+ known whether this occurs directly by a Ca -dependent phosf,hatase such + as calcineurin, or via indirect pathways involving other Ca -dependent enzymes. Phosphorylation of Ser/Thr residues in PTPs may prove to be of general significance with regard to regulation of members of this family. PTP activity in membranes of CV-l kidney cells is stimulated following treatment of intact cells with cAMP analogues to stimulate PKA, phorbol esters to stimulate PKC, or Ser/Thr phosphatase inhibitors such as okadaic acid (Brautigan & Pinault 1991). Whether this increase in activity results from direct phosphorylation of a PTP is unclear. However, PTP1 B is phosphorylated on Ser residues in HeLa cells in vivo (Frangioni et al 1992). Interestingly the PTPlB phosphorylation state is altered in a cell cycle-dependent manner, with enhanced phosphorylation observed in mitotic cells. Furthermore, im-


476

CHARBONNEAU & TO N KS

munoprecipitates of PTPIB contain a co-precipitating Ser/Thr kinase that recognizes histone HI as substrate; in mitotic cells there are up to fivefold higher levels of kinase associated with PTPI B as compared to asynchronous cell populations (N. K. Tonks et aI, submitted). The existence of such a complex suggests a novel link between steps in signal transduction pathways involving Tyr and Ser/Thr phosphorylation. PTPIB can be phosphorylated in vitro on Tyr residues to high stoichiometry by the PTK, v-abl (N . K. Tonks et aI, unpublished observations). Similar observations have been reported for LAR (Pot et al 1991). Tyrosine phos phorylation of CD45 is induced in Jurkat cells upon treatment with phytohe magglutinin or anti-CD3 antibodies, which suggests that it may occur during T-cell activation (Stover et al 1991). It is difficult to assess the effects of tyrosine phosphorylation on activity because the enzyme must be completely inhibited to prevent autodephosphorylation. Although the function of this transient phosphorylation remains unknown, it may be a manifestation of the interactions between CD45 and src-like kinases that are postulated to occur during lymphocyte activation.

Role of Targeting Domains in PTPs A general theme that is becoming apparent from consideration of the structures of the nontransmembrane PTPs is that the noncatalytic segments of these enzymes may serve a regulatory role by targeting the protein to particular subcellular locations, or modulating activity directly . Such a concept has been verified for PTPIB in which the hydrophobic segment at the extreme C-terminus of the protein is necessary and sufficient for targeting the enzyme to the endoplasmic reticulum (Frangioni et aI1992). Similarly when the 48-kd form of TCPTP is expressed in BHK cells, it is found associated with the particulate fraction of cellular extracts, which requires detergent treatment for solubilization (Cool et al 1990). The activity of this full-length form of the enzyme, as measured in extracts with reduced carboxamidomethylated and maleylated (RCM) lysozyme as substrate, is repressed. Limited trypsinization of the lysate, which removes the C-terminal segment of the PTP, is required to manifest activity . When a truncated form of 37 kd, in which the C-terminal segment was deleted by insertion of a premature stop codon, is expressed in these cells, the enzyme is constitutively active and no longer predominantly particulate (Cool et al 1990). Both 48 and 37 k forms of TCPTP have been expressed in Sf9 cells and purified to homogeneity (Zander et aI 1991). Again the full-length enzyme displayed a low level of activity against RCM lysozyme that was stimulated by limited trypsinization. Interestingly, the full-length enzyme was also activated by myelin basic protein (MBP), and PTyr-MBP is a potent substrate in the absence of trypsinization . Gel filtration analysis of lysates of BHK cells overexpressing TCPTP, or cells in which PTPIB and

1002 PROTEIN PHOSPHATA SES?

477

TCPTP are normally expressed, indicates that the full-length proteins are recovered in high Mr complexes. Efforts are now focusing on identifying the proteins with which these and other PTPs may interact in vivo and ascertaining the regulatory significance of such interactions.

PHYSIOLOGICAL FUNCTION OF PTPS


The Role of CD45 in Lymphocyte Activation Significant progress has been made toward understanding the role of CD45 in lymphocyte activation (reviewed by Shaw & Thomas 1991). CD45 is unique because little is known about the physiological role of other receptor PTPs; however, it is likely that what has been learned about CD45 will provide insight into the biological function of other receptor isoforms. When T or B lymphocytes encounter antigen, they are induced to proliferate and to eventually differentiate into mature cells capable of mediating the immune response; this process is commonly referred to as lymphocyte activation. For both T and B lymphocytes, the structure and organization of the major cell-surface receptors that recognize and bind antigen are well characterized. In contrast, little is known about the pathways by which signals generated upon antigen binding are transduced across the membrane and are eventually transmitted to the nucleus. As outlined below, CD45 has an essential role in the early signaling events of lymphocyte activation. For T-cells, it is the T-cell receptor (TCR)/CD3 complex of proteins that provides the primary mechanism for recognizing antigens carried by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. The TCR comprises the clonotypic a-13 heterodimers that are primarily involved in antigen recognition, and the tightly, but noncovalently-associated, CD3 complex that is made up of E-,,!, E-�, and �-� dimers (for review, see Clevers et al 1988). An early signaling event following stimulation of the T-cell receptor by antigen is the activation of phosphatidylinositol (PI) turnover to generate inositol trisphosphate (IP3) and diacylglycerol (DAG). Increases in IP3 and DAG result in an elevated concentration of intracellular Ca 2+ and activation of protein kinase C. These latter two events are thought to be sufficient to trigger additional signaling pathways that ultimately lead to lymphokine secretion, T-cell differentiation, and proliferation (for review, see Altman et al 1990). One of the earliest biochemical changes observed following TCR stimula tion is an increase in the state of tyrosine phosphorylation of multiple cellular proteins, including the zeta chain of the CD3 complex (for review, see Sefton & Campbell 1991). Considerable evidence now indicates that this increase in protein tyrosine phosphorylation precedes and is a prerequisite for phos 2+ pholipase C(PLC)-mediated PI turnover and elevation of intracellular Ca


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concentration (Mustelin et al 1990; June et al 1990). Moreover, Weiss et al (1991) have shown that TCR stimulation induces tyrosine phosphorylation of PLCyl . These observations suggest that TCR activation is coupled to PI hydrolysis via a mechanism involving activation of a PTK. Precedent for this type of mechanism is provided by the epidermal growth factor (EGF)-receptor kinase, which is thought to activate PI turnover by direct tyrosine phosphor ylation of PLC,), (Nishibe et al 1990) . With the TCR, control of PLC,), must be indirect since no component of the complex is known to have intrinsic protein kinase or any other enzymatic activity. Progress in elucidating the role of CD45 in these events has come primarily + from the use of mutant T-cell lines that do not express CD45. Mouse CD4 and CD8 + T-cell clones that were deficient in CD45 displayed markedly diminished capacity to proliferate upon TCR stimulation (Pingel & Thomas + + 1989; Weaver et al 1991). With both subsets of CD4 and CD8 T-cells, + reversion to the CD45 phenotype restored the ability to respond to antigen. These observations indicated that the dephosphorylation of at least one phosphotyrosine-containing protein was an essential step leading to activation. This work was extended by Koretzky et al (1990) who clearly demonstrated that CD4S- human leukemic T-cells were unable to couple TCR activation

to PI hydrolysis. In subsequent studies using CD4Y Jurkat cells, Koretzky et al (1991) detected a pronounced reduction in the extent of tyrosine phosphor ylation in response to TCR stimulation, which indicates that CD45 is essential for coupling the TCR to one or more PTKs. These findings suggest a model for T-cell activation via the TCR in which dephosphorylation by CD45 activates PTK(s) that in tum enhances PI turnover by stimulating PLC (Koretzky et al 1991 ; Weiss et al 1991). Given the fact that PTPs are capable of reversing the action of PTKs, the finding that CD45 is required for increased tyrosine phosphorylation presents a paradox. However, the regulatory features of PTKs of the src family provide a mechanism whereby a dephosphorylation reaction could ultimately lead to enhanced tyrosine phosphorylation. Members of the src family of kinases are

inhibited by phosphorylation of a conserved carboxyl-terminal tyrosyl residue (Bolen 1991). Thus, CD45 catalyzed dephosphorylation of this inhibitory site could activate src-like kinases and increase the extent of phosphorylation of T-cell proteins . If CD45 acts in this manner, then one would expect a relatively high stoichiometry of phosphorylation at the inhibitory site within target kinase(s) of the resting T-cell . As noted previously (Tonks & Charbonneau 1989), if it is to effectively regulate a src-like kinase in this manner, CD45 " cannot simultaneously dephosphorylate kinase substrates. However, there are several mechanisms by which this may be accomplished. Following dephos phorylation of its target kinase, the PTP activity of CD45 could be down-reg ulated by a feedback mechanism (e. g . phosphorylation), or CD45 and the


1002 PROTEIN PHO S PHATASE S ?

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potential kinase substrates could be physically separated by translocation to distinct cellular compartments. Interestingly, the redistribution of an intracel lular pool of CD45 has been reported to accompany T-cell activation (Minami et al 1991). Alternatively, those proteins serving as substrates of the src-like kinase may not be dephosphorylated by CD45. Which, if any, of src-like kinases is responsible for the rapid increase in tyrosine phosphorylation observed upon antigenic stimulation is not known, but the lymphocyte-specific fyn and lck kinases are generally viewed as likely candidates. The T-cell-specific kinase p56lck is associated with the intracel lular domains of the transmembrane proteins CD4 or CD8 (Rudd et al 1988; Veillette et al 1988); there is one report that this interaction is necessary for proper response to TCR stimulation (Glaichenhaus et al 1991). Several lines of evidence suggest that the inhibitory phosphotyrosine at residue 505 of p561ck may be a substrate of CD45: (a) dephosphorylation (in vitro) of the regulatory site at Tyr505 by CD45 stimulates kinase activity (Mustelin & Altman 1990), (b) the extent of phosphorylation of Tyr505 is enhanced in CD4Y lymphoma cell lines (Ostergaard et al 1989), and (c) a complex containing CD45, p561C\ and an unidentified 32-kd phosphoprotein has been isolated by immunoprecipitation (Schraven et al 1991). Despite these intri guing findings, it should be emphasized that there is as yet no definitive evidence that CD45 dephosphorylates and activates p561ck during the early stages of TCR-mediated signal transduction. Another src family kinase, p59fyn, is thought to interact directly with the T-cell receptor (Samelson et aI1990). Cooke et al (1991) have used transgenic mice that overexpress p59fyn to obtain evidence that this kinase is directly involved in TCR stimulation. An augmented response of thymocytes to TCR stimulation was observed in transgenic mice overexpressing p59fyn, but not in animals overexpressing p56lck, or an inactive mutant. At present it is not known whether the regulatory site of p59fyn is dephosphorylated by CD45 either in vivo or in vitro. In many respects, the role of CD45 in B lymphocyte activation resembles that in T-cells. Stimulation of the antigen receptor complex of B-cells, membrane IgM or IgD, increases tyrosine phosphorylation, activates protein kinase C, and elevates intracellular Ca2 + concentration (DeFranco 1987); these events initiate a cascade leading to cell proliferation. Studies employing CD4Y B-cell mutants demonstrate that CD45 expression is necessary for mobilization of intracellular Ca2 + in response to receptor stimulation (Juste ment et al 1991). These studies also provided evidence that CD45 may interact with the B-cell antigen receptor and dephosphorylate its tightly associated subunits (Justement et al 1991). To date, there is no data on the possible function of CD45 in other hematopoietic cells. Given its abundance on the cell surface and its widespread expression among all nucleated hematopoietic

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cells, it seems likely that CD45 will not only participate in lymphocyte activation, but will have additional functions in these and other cells.


Potential Role of PTPs in Cell Adhesion The transmembrane glycoproteins responsible for specific cell-cell adhesion include at least two distinct families: the cadherins, which are dependent upon 2+ Ca for their interactions (Takeichi 1991), and the N-CAM (neural cell adhesion molecule) family, which is a member of the immunoglobulin (Ig) 2+ superfamily and is Ca -independent (Edelman & Crossin 1991). Proteins of the cadherin and N-CAM families both function primarily through homophi lic-binding mechanisms (i.e. they bind identical molecules on the surface of adjacent cells). Biochemical and genetic analyses of these cell adhesion molecules indicate that their function may not be restricted to simple recognition and mechanical adhesion, but may also include activation of intracellular signaling pathways linking external cell contacts to alterations in intracellular processes such as gene expression, cytoskeletal organization, and cell division. Doherty et al (1991) found that phenotypic changes (e.g. neurite outgrowth) in PC12 cells were induced by cell-cell contacts mediated by N-CAM and N-cadherin. The N-CAM and N-cadherin-induced phenotypic changes were accompanied by an influx of Ca2 + that was dependent on a pertussis toxin-sensitive activation 2+ of two types of Ca channels. This confirmed previous work by Schuch et + al (1989) who found that antibodies to N-CAM or Ll also activated Ca2 channels via a pertussis toxin-sensitive mechanism. The resemblance between the extracellular domain of LAR (Streuli et al 1988) and that of the N-CAM family of cell adhesion molecules provided the first indication that PTPs may be involved in homophilic cell-cell interactions. The configuration of Ig-like and FNIII repeats within the external domains of LAR and the related type II receptor PTPs (see above) are very similar to those of the N-CAMs, but the PTPs typically have fewer Ig-like domains and more FNIII repeats. In view of these structural similarities, it is reasonable to postulate that type II receptor PTPs may constitute a new group of cell adhesion molecules with recognition and binding properties analogous to those of N-CAM, but with internal domains equipped to transduce signals arising from cell-cell contact by altering the extent of tyrosine phosphorylation of their target proteins. Unlike N-CAMs and N-cadherins, which appear to be coupled to second messengers via indirect mechanisms, the PTPs could respond to external ligands with a direct signal. HPTP�and several other type III receptor PTPs are also of interest in this regard since their external domains mainly comprise tandem FNIII-like repeats. Some type III repeats within fibronectin are involved in binding to cells via integrin receptors or binding


1002 PROTEIN PHO SPHATASES?

48 1

heparin. It is conceivable that type III receptor PTPs could also be involved in cell adhesion events, perhaps by heterophilic binding of ligands on adjacent cells, or by interacting with components of the extracellular matrix such as heparin. Interaction between the boss/sev gene products in the development of ommatidia in the Drosophila retina may serve as a paradigm for the role of receptor PTPs in mediating cell adhesion. The sevenless (sev) gene of Drosophila is required for differentiation of precursor cells into the R7 photoreceptor cell of the mature retina (Rubin 1989). The sev gene encodes a large PTK with two predicted transmembrane segments, a single extracellular loop (200 kd) containing seven FN-Ill-like repeats, and a cytoplasmic kinase domain that is required for biological function (Norton et aI 1 990). The ligand for sev is a membrane glycoprotein (120 kd) containing seven transmembrane segments, which is encoded by the boss gene. The boss gene product, which is expressed in adjacent R8 celis, supplies the inductive signal to R7 by binding to sev and apparently modulating its PTK activity in a process requiring direct cell-cell contact (Kramer et al 1991). Speculation about probable ligands and functions of these receptor PTPs must be made with caution. In addition to N-CAMS, FNIII-like domains are present in a wide variety of receptors that bind relatively small polypeptide ligands; these include cytokine (IL6, IL4, and IL2) , growth hormone, prolactin , interferon, and erythropoietin receptors (B azan 1 990; Patthy 1 990). FNIII-like motifs have also been identified in the external segments of the insulin and insulin-like growth factor receptors, and in a related group of receptor PTKs including trk, eck, elk, eph, and met (O'Bryan et al 1991). Furthermore, twitchin, an intracellular kinase from C. elegans, also contains a large segment with FNIII and Ig-like repeats (Beniam et aI 1 989) . Interestingly, N-CAM-like extracellular domains are located within the extracellular segments of two newly discovered receptor tyrosine kinases designated ark and axl (Rescigno et al 199 1 ; O' Bryan et al 1 991 ). Perhaps the strongest data in support of a role for PTPs in cell adhesion and development come from recent studies of three receptor isoforms from Drosophila; DLAR, DPTP lOD, and DPTP99A (Yang et al 1 99 1 ; Tian et al 199 1 ; Hariharan et al 1991). Immunocytochemistry and in situ RNA hybridization demonstrated that the expression of all three PTPs was restricted to central nervous system axons where both the timing and pattern of their appearance was consistent with a role in regulating neurite outgrowth and pathfinding during development. Obviously, much remains to be done to establish a role for these and other receptor PTPs in cell adhesion. Significant advancements will come with identification of ligands and physiological substrates.

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PTPs and the Control of the Timing of Mitosis Over the past four years there has been a remarkable synthesis of three distinct lines of research into a single, coherent picture of the control of the transition from G2 to M phase of the eukaryotic cell cycle (reviewed in Murray & + Kirschner 1989). In primarily genetic studies the product of the cdc2 gene in S. pombe (CDC28 in S. cerevisiae) was recognized as an essential requirement for progression through both the G l iS and G2/M transitions of the cell cycle. Parallel biochemical studies in clam and sea urchin eggs identified a class of proteins that accumulate during interphase, but are degraded abruptly at the metaphase:anaphase transition of mitosis. These proteins are termed cyclins . The link was provided by the biochemical characterization of maturation-promoting factor (MPF) from Xenopus 00cytes. These oocytes are physiologically arrested in prophase I of meiosis , but can b e induced to mature to unfertilized eggs, arrested at second meiotic metaphase, by treatment with progesterone or insulin. Injection of egg cytoplasm, which contains MPF, into oocytes also induced the maturation response . MPF has now been identified in mitotic cells from yeast to humans and is most likely responsible for driving the G2/M phase transition in all d2 eukaryotic cells. Purified preparations of MPF were shown to contain p34c C and cyelin. + CdC2 The cdc2 gene product, p34 , is a protein Ser/Thr kinase, the activity of which is regulated by changes in its phosphorylation state and alterations in its association with various regulatory molecules. In G1 phase of the cell cdc2 is an inactive monomer. There is evidence to suggest that it cycle p34 may be phosphorylated on Ser277 at this point (Krek & Nigg 1991). Experiments utilizing Xenopus e1c¥ extracts indicate that binding to cyclin C on three sites, Thr l 4 and Tyrl S, which induces phosphorylation of p34c are inhibitory, and Thr1 61 (equivalent to Thr1 67 in S. pombe), which is an activating modification (Solomon et al 1992). The adjacent Thrl 4 and Tyrl S residues are located within the sequence GXGXXG, the putative nucleotide binding pocket for the kinase and are presumably inhibitory through interfer cdc2 / ence with binding of ATP. At a critical threshold concentration of p34 cyclin at the G2/M transition, there is a decrease in the rate of phosphorylation of these sites and an increase in the rate of their dephosphorylation (Solomon cdc2 as a et al 1990). This provides a highly sensitive switch to activate p34 histone H I kinase. The phosphorylation of Thrl 6 1 catalyzed by a partially characterized kinase termed cak (Solomon et al 1992) persists into mitosis and is essential for the maintenance of kinase activity. Its phosphorylation is countered by a molecule termed INH, a negative rcgulator of MPF activation, which is a form of protein phosphatase 2A, a Ser/Thr phosphatase (Lee et al 1991). Proteolytic destruction of the associated cyclin is an important step in cdc2 the deactivation of p34 for return to interphase. However, the details of


483

the trigger and the mechanism by which this is achieved, coupled with the role played by SerlThr phosphatases , remain to be described in detail .

The timing o f mitosis i n S. pombe i s determined b y the balance between

the activity of an inhibitory pathway involving the weel + gene product and

an activatory pathwa involving the product of the cdc25 + gene, both acting 4clcc2 at the level of p 3 . Wee l is a member of the growing family of dual specificity kinases that in structure most closely resemble Ser/Thr kinases,


but possess the ability to phosphorylate tyrosyl residues (Lindberg et aI 1992). 4 Its only known target is p 3 cdc2:cyclin. It is responsible for phosphorylation

of the inhibitory site at Tyrl 5 and, in higher eukaryotes , it is possibly also 4 responsible for phosphorylation of the adjacent residue, Thrl . Weel acts cooperatively with mik- l , which is another kinase (Lundgren et al 1 99 1 ) .

Furthermore the nim1 + gene product, which also possesses structural features

of a kinase, is suggested by genetic evidence to function as a negative regulator of wee 1 (Russell & Nurse 1 987) . Counteracting the effects of wee1 is the

product of the cdc2S+ gene. Gould & Nurse established that when the temperature-sensitive mutant of cdc25 , cdc25 -22 , was arrested at the restric 4cdc2 phosphory tive temperature, the cells were synchronized in G2 with p 3

lated on Tyr l S . Upon return to the permissive temperature there is 4 dephosphorylation of p 3 cdc2 , stimulation of its histone kinase activity, and 34 C2 the cells enter mitosis (Gould & Nurse 1989) . A mutant form of p cd , in which Tyr1 5 had been replaced by Phe and thus could not be phosphorylated

on tyrosyl residues , bypassed the requirement for the cdc2S+ gene product,

cdC25 . Expression of this mutant rescued a strain deleted for cdc25 and also p80 rescued growth of cdc2S-22 at the restrictive temperature (Gould & Nurse 1 989) . In both cases the cells advanced prematurely into mitosis and displayed

the wee phenotype. In addition , expression of TCPTP in cdc25-22 also rescued growth at the restrictive temperature and advanced cells into mitosis prema

turely, fcroducing effects that correlated with the dephosphorylation of Tyr 1 5 4c c2 in p 3 by the PTP (Gould et al 1990) . These data all point to an intimate

d 25 in the dephosphorylation process. But is it actually

involvement of p80c c

a phosphatase? The consensus of current data indicates clearly that it is. Initial analysis of the sequence of p80cdc25 did not detect homology with

any protein in the database, including the PTP family. However, when Guan

et al ( 199 1 a) reported that the VHl protein of the pox virus vaccinia was a phosphatase distantly related to the members of the PTP family, several grouPss detected limited similarity between the sequence of VHl and p80cdC

5

,

particularly in the signature motif, [IIV]HCXAGXXR[S/T]G , that character

izes the PTPs . A flurry of papers followed in which exceedingly low levels

of activity in preparations of cdc25 were demonstrated (Strausfeld et al 1 99 1 ;

Dunphy & Kumagai 1 99 1 ; Gautier et al 1 99 1 ; Millar et al 1 99 1 ; Lee et al

1 992). For example with pNPP (para nitrophenyl phosphate) as a substrate


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CHARBONNEAU & TONKS

cdc25 displays a turnover number of 2 min- ! as compared to 1200 sec- ! for YOP2b_ S imilarly 1 25-fold higher quantities of cdc25 , expressed in bacteria as a glutathionine-S-transferase (GST) fusion protein, than TCPTP, expressed in Sf9 cells using the baculovirus system, were required for similar rates of dephosphorylation of pNPP (Millar et aI 1 99 1 ). Perhaps the most compelling evidence for a phosphatase function of cdc25 is the demonstration that mutations in the PTP signature motif, specifically Cys to Ala or Ser, and Arg cdc2 in vitro, induce to Lys or Met, abrogate its capacity to dephosphorylate p3 4 maturation in Xenopus oocytes, and rescue the temperature-sensitive cdc25-22 mutation in S. pombe (Gautier et al 1 99 1 ). In addition cde25 is sensitive to inhibitors of PTPase activity , ineluding thiol-direeted reagents (Dunphy & Kumagai 1 99 1 ). Together these data support the conclusion that although the activity in vitro is low, and unlike other PTPs appears to be highly spe ede cific for its particular substrate p 3 4 2 , edc25 , nonetheless, is a protein phosphatase. d2 One explanation for the discrimination of p80ede25 towards p34 e e :cyelin complexes is that the eyelin is a multifunctional protein activating both ee ede2 and p80 d 25 . Galaktionov & Beach ( 1 992) noted that in PTPs such p34 as PTP l B the signature motif for members of the family is adjacent to a segment of similarity to cyelins. The similarity is detected with the sequence at the junction of the eyelin box, notably in cyelin B. While cdc25 contains the signature sequence, it does not have the cyelin-related sequence (Figure 2). These investigators demonstrated that addition of cyelin B to bacterially expressed cdc25 stimulated its activity fourfold against a variety of artificial substrates including ReM lysozyme. Activation was most pronounced when cyelin B and cdc25 were added in stoichiometric amounts. Furthermore , a stable association of cdc25 with p3 4cde2 :eyelin B in vivo was demonstrated. These data suggest a model wherein the catalytic segment of the PTP, such as PTPI B , is activated by a cis-acting cyelin-related motif situated C-terminal to the active site cysteine in the polypeptide chain. In cdc25 this activating motif is provided in trans by association with cyelin. Site-directed mutagenesis studies to test this hypothesis further are underway. While cdc25 remains the PTP for which the physiological substrate has been best defined, the picture is rapidly becoming more complicated. Now at least three human cdc25 genes have been identified and the families of cdc2-like kinases and cyelins are also expanding rapidly. Electrophoretic mobility shifts, consistent with phosphorylation, have also been observed in cdc25 with the suggestion of a feedback phosphorylation of the phosphatase d by p3 4e e2 (Kumagai & Dunphy 1992). This and the activation of cdc25 by eyelin B may explain the observed autoaetivation of pre-MPF. In higher eukaryotes, where both Thr l 4 and Tyr 15 are phosphorylated, the identity of the Thrl 4 phosphatase remains to be formally established although this is


A

ill

CA V

Consensus cdc25A

I

HC

TDGKRV

cdc25B cdc2SC

R FSSE

P

FSSE

P

E'SSE

GP

stg

F'5SE

25Sp

HS

:!

YVRERDRLGNE _ - YPKLHYPEL

GGYKEFFMKCQSYCEPPSYRPMHHE

F l RERDRAVND--YPSLYYPEMYI

Protein tyrosine phosphatases.

Targeting subunits for protein phosphatases.

Protein phosphatases in pancreatic islets.

Protein phosphatases in cell signalling.

threonine phosphatases; an expanding family.

Intrasteric regulation of protein kinases and phosphatases.

threonine phosphatases.

Receptor-type protein tyrosine phosphatases in cancer.

Protein phosphatases possibly involved in rat spermatogenesis.

Protein tyrosine phosphatases as potential therapeutic targets.

When worlds collide: immunosuppressants meet protein phosphatases.

Hepatic protein tyrosine phosphatases in the rat.

Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research.

Substrate specificity and plasticity of FERM-containing protein tyrosine phosphatases.

Protein kinase and phosphatases from human polymorphonuclear leucoytes.

Characterization of neuronal protein phosphatases in Aplysia californica.

The structure, role, and regulation of type 1 protein phosphatases.

The regulation and function of protein phosphatases in the brain.

A Drosophila-centric view of protein tyrosine phosphatases.

Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

Cellular biochemistry methods for investigating protein tyrosine phosphatases.

Genetic alterations of protein tyrosine phosphatases in human cancers.

Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation.

Protein tyrosine kinases and phosphatases in the nervous system.