A MAPK docking site, distinct from the phosphoacceptor site, was first identified in c-Jun 89, a c-Jun N-terminal kinase (JNK) substrate; this site was designated the "δ domain". Subsequently, a JNK binding site in the transcription factor ATF-2 1011 and a motif termed the "d-box" of Elk-1 that binds ERK2 412 were noted to be similar in sequence to the JNK binding site in c-Jun. Related motifs have been identified in a number of other proteins and have been given various names, including DEJL (docking sites for ERK and JNK, LXL) domain 4, kinase interaction motif (KIM) 1314, MAPK-docking site 1516, D box 512, D-site 17 and D-domain 6181920. It is important to note that, although these domains were identified based on the ability to bind one or more MAPK, there are differences in the consensus sequences used to identify each of them. For example, MacKenzie et al. 14 proposed a consensus KIM sequence of (V/L)X₂(R/K)(R/K)X_(3–6)L, with V, L, R, and K representing the amino acids valine, leucine, arginine and lysine, respectively; Bardwell et al. 16 define a consensus MAPK-binding site sequence of (R/K)₂X_(2–6)(L/I)X(L/I), with I representing the amino acid isoleucine; and Kornfeld and colleagues 4 reported two consensus sequences for the DEJL domain: (K/R)X(X/K/R)(K/R)X_(1–4)(L/I)X(L/I) and (K/R)(K/R)(K/R)X_(1–5)(L/I)X(L/I). In the present studies we use the term D-domain and the consensus sequences reported by Kornfeld and colleagues 4.

Sharrocks and colleagues 21 report that D-domains are characterized by a cluster of basic residues positioned amino-terminal to an (L/I)X(L/I) motif followed by a triplet of hydrophobic amino acids that precedes a series of proline residues 1721. These investigators assessed the role of each of these regions in the binding of ERK2 and p38 to transcription factors, MEF2A, SAP-1, and Elk-1. They determined that mutation of the basic region of the transcription factors reduced their phosphorylation by both phospho-ERK2 and phospho-p38 21. This suggests that the basic residues are important for both ERK2 and p38 targeting of MAPK substrates. Mutation of the (L/I)X(L/I) motif (also called the LXL motif) diminished phosphorylation of phospho-ERK substrates, whereas it is not required for phosphorylation of substrates by the MAPK, phospho-p38 21. It was also determined that the hydrophobic patch plays an important role in phosphorylation of the substrates by both phospho-ERK and phospho-p38; however, this patch is more important for p38 binding than ERK2 binding. Barsyte-Lovejoy et al. 21 concluded that the proline residues were not important in specificity determination of MAPK substrates. Therefore, the authors hypothesize that the proline residues may play a structural role within the motif.

D-domains can show specificity for families of MAPKs; for example, the Elk-1 D-domain binds JNK and ERK, but not p38 522; both the SAP-1 and Elk-1 D-domains bind ERK2, whereas the SAP-1, but not Elk-1, D-domain binds p38α 22. Other D-domains show specificity within a MAPK family; for example, the SAP-1 D-domain binds p38α and p38β, but not p38δ 22. The D-domain can be positioned either N- or C-terminal to the phosphorylation site 712161923.

A second MAPK docking motif has also been identified: the FXFP motif, or DEF (docking site for ERK, FXFP) motif 41822, where F and P represent the amino acids phenylalanine and proline, respectively. Binding and substrate phosphorylation can occur in the absence of the proline residue 1418; however, its presence does increase the effectiveness of the motif 18. Thus, we chose to include the proline in our searches. The identity of the second (X) residue is highly variable 418. In most, if not all, proteins, the FXFP motif is C-terminal to the phosphorylation site 4181924. In general, it appears that the FXFP motif occurs more proximal to a phosphorylation site than is often the case for a D-domain 418202224. The FXFP motif binds ERK2 and p38α 1822, but not JNK3 4 and p38β 422. The FXFP motif and D-domain are each sufficient for MAPK docking; however, when both are present in a protein, they function additively 1822.

We recently identified a D-domain sequence in PLC-γ1 and provided strong evidence that this sequence mediated an observed interaction between PLC-γ1 and phospho-ERK2 25. We have also reported that PLC-γ2, -β1, -β2, and -β4 each have at least one identifiable D-domain, as well co-immunoprecipitate with ERK2 26. Based on these observations, we have proposed that MAPK signaling and the metabolism of PIs are integrated. In order to substantiate this hypothesis, we sought to determine whether MAPK docking sites could be recognized in other enzymes that metabolize PIs; additionally, we sought support for extending this hypothesis to include the metabolism of IPs.

Eight PIs and more than 20 IPs have been identified 27282930. Several reviews of the metabolism and cellular roles of these molecules have appeared 272829303132333435. As the physiologic functions of the PIs and IPs were not a primary focus of the present studies, we will not summarize this information here; instead, the interested reader is directed to the sources cited above; we acknowledge that this is only a partial listing of the reviews that have been written on these subjects. PIs are substrates for a variety of phospholipases, acyl transferases, kinases and phosphatases, while IPs are metabolized by a series of kinases and phosphatases. Of these enzymes, we have limited the scope of the present studies to kinases and phosphatases. In order to assist the reader in understanding the reactions catalyzed by the enzymes which we analyzed, the pathways for the metabolism of PIs and IPs by various kinases and phosphatases are shown in Figures 1A and 1B, respectively. We use the IUPAC-IUB nomenclature for the identification of phosphatidylinositol (PtdIns) and inositol (Ins) phosphates (P): numbers are used to designate the carbon atoms to which phosphate groups are bound and the total number of phosphate groups is designated by a subscript, with the exception that no subscript is employed to designate the presence of a single phosphate group. For example, phosphatidylinositol 4-phosphate is designated PtdIns4P and inositol 1,4,5-trisphosphate is designated Ins(1,4,5)P₃. For a review of inositol phosphate chemistry the reader is referred to recent articles by Shears 36 and Irvine 37.

We obtained the primary sequences of human and mouse kinases and phosphatases that control the phosphorylation state of the inositol ring in PIs and IPs from GenBank at NCBI and searched these sequences for an FXFP motif and the two consensus sequences for a D-domain reported by Kornfeld and colleagues 4: (K/R)X(X/K/R)(K/R)X_(1–4)(L/I)X(L/I) or (K/R)(K/R)(K/R)X_(1–5)(L/I)X(L/I). We note that this strategy most likely failed to identify all possible sequences that may function as MAPK docking sites. For example, both the human and mouse Class I PI 3-kinase β include the sequence LILRRHGNLFI, which contains a KIM, as defined by MacKenzie et al 16, and a MAPK-docking site, as defined by Bardwell et al. 16, but not a D-domain according to the criteria that we employed. Similarly, the human and mouse Ins(1,3,4)P₃ 5/6-kinase/Ins(3,4,5,6) 1-kinase contain the sequence LCRKRGXEVVQLNL (X is M in human and I in mouse), which fits the consensus sequences for a KIM and a MAPK-binding site, but not a D-domain. We chose to use the criteria of Kornfeld and colleagues based on its successful application in the identification of D-domains in PLC isozymes 2526. Although the list that we have compiled is likely to be incomplete, it does serve as a useful first approximation. We also searched each of the enzyme sequences for potential MAPK phosphorylation sites (i.e., (S/T)P sequences) and MAPK optimal phosphorylation sequences, PX(S/T)P. Finally, we note that we did not analyze the sequences of enzymes that control the metabolism of the diphosphorylated IPs.

In order to facilitate the presentation of the data, we separated human from mouse enzymes, kinases from phosphatases, and enzymes in which we identified a D-domain and/or FXFP motif from those in which we did not. Human and mouse kinases having a D-domain and/or FXFP motif are contained in Tables 1 and 3, respectively, whereas human and mouse kinases that do not have either of these sequences are presented in Tables 2 and 4, respectively. Similarly, human and mouse phosphatases containing a D-domain and/or FXFP motif are listed in Tables 6 and 8, respectively, while human and mouse phosphatases that are devoid of these sequences are contained in Tables 7 and 9, respectively.

In the following discussion we use the terms "alternative pair" and "overlapping" in referring to relationships of D-domains. An alternative pair of D-domains has the same amino-terminus and two possible carboxyl-termini: e.g., ¹⁵RRRDAVIAL and ¹⁵RRRDAVIALGI in human PI 4-kinase α (Table 1). We use the term overlapping to identify D-domains that have distinct amino-termini and carboxyl-termini, but share a region of sequence: e.g., ³⁷⁶RNSKGERLLL and ³⁷⁹KGERLLLYI found in human PI4P 5-kinase type Iα (Table 1).

Phosphatidylinositol 3-kinase (also called phosphoinositide 3-kinase, PtdIns 3-kinase, PI 3-kinase, and PI3K) isozymes catalyze the phosphorylation of the 3-position of the inositol ring of phosphatidylinositols 32. Three classes (I, II, and III) of PI 3-kinase have been identified 3839. These classes are differentiated on the basis of their subunit composition, substrate specificity and mechanisms of regulation. The Class I enzymes are heterodimers of a regulatory subunit, of various sizes, and a catalytic subunit of approximately 110 kDa. Three distinct forms of the catalytic subunit (p110α, p110β, and p110δ) and five forms of the regulatory subunit (p85α, p85β, p55α, p55γ, and p50α) have been identified. The class IB isoform consists of a p101 regulatory subunit coupled to a p100γ catalytic subunit. Class I enzymes catalyze the synthesis of PtdIns3P, PtdIns(3,4)P₂, and PtdIns(3,4,5)P₃ 4041; PtdIns(4,5)P₂ may be the preferred substrate in vivo 42. Class II isozymes are monomeric catalytic subunits (α, β, and γ) containing a carboxyl-terminal C2 domain; these enzymes may be referred to as PI3K-C2. PtdIns and PtdIns4P, and under certain conditions PtdIns(4,5)P₂, are substrates for the Class II PI 3-kinase 3943. A single Class III isozyme, which is specific for PtdIns, has been identified 44.

The Class I p110α and p110γ human (Table 1) and mouse (Table 3) proteins contain D-domains, whereas the p110β (Tables 2 and 4) and p110δ (Tables 1 and 4) isozymes do not. Of all the PI 3-kinase sequences that we analyzed, only the human PI 3-kinase δ (Table 1) contains an FXFP motif (⁵⁸⁵FSFP).

The Class II PI3-kinase C2-α contains a D-domain that is conserved in the human (Table 1) and mouse (Table 3) isozymes. PI 3-kinase C2-α was the only PI 3-kinase isozyme in which we found an optimal MAPK phosphorylation sequence (Tables 1, 2, 3, 4). The human and mouse PI 3-kinase C2-α proteins each contain three such sequences; a sequence alignment revealed that two of these (PLTP and PATP) are conserved, while the third, ¹¹⁸PVTP and ¹³⁷⁴PFSP in human and mouse, respectively, is unique. Both the human (Table 1) and mouse (Table 3) PI 3-kinase C2-γ contain D-domains. A sequence alignment of these two proteins revealed that only the last D-domain (¹³⁴⁷KHMKNIHL in human and ¹⁴⁰⁹KHLKNIHL in mouse) aligns; all of the other identified D-domains are unique to one or the other protein. These results indicate the MAPK-dependent regulation of the human and mouse Class II PI 3-kinases may be significantly different and, thus, caution should be exercised when comparing studies on these two proteins.

The human (Table 1) and mouse (Table 3) Class III PI 3-kinase contains a conserved D-domain. Neither of these proteins contains an FXFP motif or an optimal phosphorylation sequence for MAPKs.

Phosphatidylinositol 4-kinase (PtdIns 4-kinase, PI 4-kinase) catalyzes the phosphorylation of PtdIns to produce PtdIns4P. Subfamilies (Types II and III; or PI4K230, PI4K92 and PI4K55 using the nomenclature of Heilmeyer et al. 45) of PI 4-kinase have been identified; these posses a conserved C-terminal catalytic domain and diverse N-terminal regulatory domains 45. Differing physiologic functions have been ascribed to each of the three subfamilies 4546474849.

PI4Kα (also called PI 4-kinase 230) contains three alternative pairs of D-domains that are conserved in human (Table 1) and mouse (Table 3) sequences. PI4Kα also contains an FXFP sequence and two optimal MAPK phosphorylation sequences that are conserved in human and mouse proteins. PI 4-kinase β (also called PI 4-kinase 92) contains three D-domains that are conserved in the human (Table 1) and mouse (Table 3) isozymes. The first of these (²³²RGTKLRKLIL) overlaps with an identifiable bipartite nuclear localization sequence motif 45. The human and mouse PI 4-kinase-β do not contain an FXFP motif or optimal MAPK phosphorylation sequence. The human (Table 1) and mouse (Tabl3 3) PI 4-kinase type II (also called PI 4-kinase 55) contain a conserved D-domain and optimal phosphorylation sequence for a MAPK. Neither protein contains an FXFP motif. The human (Table 1), but not the mouse (Table 4), PI 4-kinase type II-β contains a single D-domain, while neither protein contains a sequence fitting the MAPK optimal phosphorylation sequence or the FXFP motif.

Phosphatidylinositol phosphate kinases (PIP kinases, PIPKs) utilize PtdIns3P, PtdIns4P, and PtdIns5P as substrates, catalyzing the synthesis of PtdIns(3,4)P₂, PtdIns(3,5)P₂, and PtdIns(4,5)P₂ (Table 5). These enzymes are also able to catalyze the formation of PtdIns5P and PtdIns(3,4,5)P₃ (Table 5). Three types (I, II and III) of PIP kinase are defined based on primary sequence, substrate specificity, and subcellular localization 51. These families are often designated by the reaction that they catalyze most efficiently. Thus, the Type I PIP kinases are also termed PI4P 5-kinase, the Type II PIP kinases are termed PI5P 4-kinases, and the Type III PIP kinases are called PI3P 5-kinases. Three forms (termed α, β, and γ), each with multiple splice variants, of Type I and Type II PIP kinase have been identified 52535455. It should be noted that the nomenclature for the α- and β-forms of the human and mouse Type I PIP kinase are reversed: that is, the human Type Iα enzyme corresponds to the mouse Type Iβ enzyme, and vice versa.

Each of the Type I isozymes contain at least one identifiable D-domain that was conserved in humans and in mice. In human PI4P 5-kinase Type Iα (Table 1) an alternative pair of D-domains (³⁷⁹KGERLLLYI and ³⁷⁹KGERLLLYIGI) overlaps with a different D-domain (³⁷⁶RNSKGERLLL). This observation raises the intriguing possibility that these sequences are specific for binding of a particular MAPK, or MAPK family, and that there is competition among MAPKs for binding, with the likelihood that a mechanism(s) exists for the regulation of this binding. Further, the MAPK that is bound may affect the kinetics of the signal that is generated and, thus, the downstream response. The identified alternative pair of D-domains in PI4P 5-kinase type Iγ (PIPKIγ) (Tables 1 and 3) may be responsible for binding phospho-ERK1, which has been shown to catalyze the phosphorylation of serine 650 in PIPKIγ 56. Phosphorylation of serine 650 inhibits the binding of talin to PIPKIγ and may play a role in synaptic neurotransmission and focal adhesion disassembly during mitosis. None of the Type I PIP kinases that we analyzed contains an FXFP motif (Tables 1 and 3). Each of the Type I PIPKs that we analyzed contains an optimal MAPK phosphorylation site that is conserved in human and mouse proteins. In the case of PIPKIγ, this is not serine-650. However, it is possible that other MAPKs are capable of phosphorylating threonine-512 (human)/threonine-511 (mouse).

The Type II isozymes (Tables 2 and 4) were found to be devoid of D-domain sequences and FXFP motifs. However, the Type IIα and Type IIβ isozymes do contain an optimal MAPK phosphorylation sequence that is conserved in human (Table 2) and mouse (Table 4) protein sequences.

The PIKfyve human (Table 1) and mouse (Table 3) sequences each contain six MAPK optimal phosphorylation sequences. This is the most that we found in any of the sequences that we analyzed. These enzymes contain three conserved D-domain sequences, but are devoid of an FXFP motif.

Ins(1,4,5)P₃ 3-kinase catalyzes the formation of Ins(1,3,4,5)P₄ from Ins(1,4,5)P₃. Three forms of Ins(1,4,5)P₃ 3-kinase have been cloned; these forms differ in their molecular mass, regulation by Ca²/calmodulin, tissue distribution and intracellular localization 5758596061626364. All three forms of human (Tables 1 and 2) and both forms of mouse (Table 4) Ins(1,4,5)P₃ 3-kinase contain one or more optimal sequence for MAPK phosphorylation. Human Ins(1,4,5)P₃ 3-kinase B contains both an FXFP motif and a D-domain, whereas the A and C isoforms do not contain either of these two MAPK docking sequences (Tables 1 and 2). Ins(1,4,5)P₃ 3-kinase B, which plays a critical role in T-cell development 6566, is associated with the endoplasmic reticulum via its N-terminus 5767. Thus, it is possible that the MAPK binding to the ⁵⁶FLFP sequence motif and/or phosphorylation of serine-71 within the optimal phosphorylation sequences (⁶⁹PRSP) regulates Ins(1,4,5)P₃ 3-kinase B interaction with the endoplasmic reticulum.

Wilson and Majerus 68 cloned an Ins(1,3,4)P₃ 5/6-kinase, which was subsequently shown by Yang and Shears 69 to be the same as Ins(3,4,5,6)P₄ 1-kinase. Interestingly, this enzyme also possesses Ins(1,3,4,5,6)P₅ 1-phosphatase activity 70. Regulation of these latter two reciprocal activities provides a mechanism for tight control of Ins(3,4,5,6)P₄ levels in cells. The production of Ins(1,3,4,6)P₄ by Ins(1,3,4)P₃ 5/6-kinase is the rate-limiting step in the synthesis of inositol hexakisphosphate from Ins(1,3,4)P₃ 71. We were unable to identify an optimal sequence for MAPK phosphorylation, an FXFP motif or a D-domain in human (Table 2) or mouse (Table 4) Ins(1,3,4)P₃ 5/6-kinase/Ins(3,4,5,6) 1-kinase.

Inositol polyphosphate multikinase catalyzes the formation of Ins(1,3,4,5,6)P₅ from Ins(1,4,5)P₃ by phosphorylation of both the 3- and 6-position of the inositol ring 3036, with phosphorylation of the 3-position possibly preceding that of the 6-position 72. The enzyme also phosphorylates the 1-position of Ins(4,5)P₂ to form Ins(1,4,5)P₃ 72. Majerus and colleagues 73 reported that in vitro the enzyme displays specificity as an Ins(1,3,4,6) 5-kinase. Neither the human (Table 2) nor mouse (Table 4) protein contains an FXFP motif, a D-domain or optimal sequence for MAPK phosphorylation.

Ins(1,3,4,5,6)P₂ 2-kinase catalyzes the final step in the synthesis of inositol hexakisphosphate from Ins(1,3,4)P₃, and ultimately from Ins(1,4,5)P₃ 71. The human 2-kinase contains an FXFP motif, but does not contain a D-domain (Table 1). We did not identify an optimal sequence for MAPK phosphorylation.

Inositol monophosphatase is a Mg²⁺-dependent enzyme that catalyzes the hydrolysis of Ins1P, Ins3P and Ins4P, as well as several related compounds, but does not hydrolyze Ins2P 7475767778. Inositol monophosphatase has received significant attention as a potential site of action for lithium in the treatment of bipolar disorder. However, recent studies have identified a number of other lithium targets, as well 79.

Three forms of inositol monophosphatase have been identified: A1 (IMPA1), A2 (IMPA2) and A3 (IMPA3). Of these three forms, only inositol monophosphatase A1 contains an identifiable D-domain (Tables 6 and 8). The human and mouse sequences differ by two amino acids in this region resulting in an alternative pair of D-domains in the mouse isoform (and ²⁶¹RIAKEIEI and ²⁶¹RIAKEIEIIPL) and a single D-domain in the human isoform (²⁶¹RIAKEIQVIPL). We did not find an optimal sequence for MAPK-catalyzed phosphorylation or an FXFP motif in any of the human or mouse inositol monophosphatase isozymes.

Inositol polyphosphate 1-phosphatase is a Mg²⁺-dependent enzyme that hydrolyzes Ins(1,4)P₂ and Ins(1,3,4)P₃, and is inhibited by lithium 76808182. Similar to inositol monophosphatase, the 1-phosphatase has received attention as a site of therapeutic action for lithium in the treatment of bipolar disorder 83. Notably, the human (Table 6), but not the mouse (Table 9), 1-phosphatase contains a D-domain. Neither the human nor mouse enzyme contains an FXFP motif or an optimal sequence for MAPK phosphorylation.

Myotubularin, which was originally identified as a candidate gene mutated in X-linked myotubular myopathy 84, has been shown to possess PtdIns3P 3-phosphatase activity 8586. A number of myotubularin-related (MTMR) proteins have also been identified 8788. Myotubularin (also called MTM1), MTMR1, MTMR2, MTMR3, and MTMR6 possess PtdInd(3)P 3-phosphatase activity 89. In addition to their PtdIns3P 3-phosphatase activity, myotubularin, 90, MTMR2 91, MTMR3 92, and MTMR6 90 also possess PtdIns(3,5)P₂ 3-phosphatase activity; however, it should be noted that Kim et al. 89 reported that myotubularin and MTMR2 do not hydrolyze PtdIns(3,5)₂. Additionally, myotubularin and MTMR2 have been shown to hydrolyze Ins(1,3)P₂ 89.

When we analyzed the human (Table 7) and mouse (Table 9) myotubularin protein sequences, we did not find either an FXFP or D-domain sequence. However, the human sequence does contain two optimal MAPK phosphorylation sequences not present in the mouse sequence. We identified a conserved D-domain in the human (Table 6) and mouse (Table 8) MTMR1 protein, but did not find an optimal sequence for MAPK phosphorylation or FXFP motif in either protein. Neither the human (Table 7) nor the mouse (Table 9) MTMR2 has an optimal sequence for MAPK phosphorylation, a D-doman, or an FXFP motif. Although the human (Table 7) and mouse (Table 9) MTMR3 and MTMR6 each contain a sequence fitting the optimal sequence for MAPK phosphorylation, they do not contain an identifiable D-domain or FXFP motif.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a PtdIns(3,4,5)P₃ 3-phosphatase 93, as well as Ins(1,3,4,5)P₄ 3-phosphatase 93 and Ins(1,3,4,5,6)P₅ 3-phosphatase 94. The human (Table 6) and mouse (Table 8) PTEN sequences contain a conserved FXFP sequence, but no identifiable D-domain or optimal MAPK phosphorylation sequence.

Two forms (Types I and II) of inositol polyphosphate 4-phosphatase have been identified 959697. These enzymes cleave the 4-phosphate from Ins(1,3,4)P₂, Ins (3,4)P₂, and PtdIns(3,4)P₂. The Type I 4-phosphatase has been reported to localize to endosomes, where it plays an important role in the generation of PtdIns3P 98. In growth factor-stimulated cells the Type I 4-phosphatase also localizes to plasma membrane ruffles, where it hydrolyzes PtdIns(3,4,)P₂, thereby regulating the association of PtdIns(3,4)P₂-binindg proteins with the plasma membrane 98. We identified a conserved D-domain in human (Table 6) and mouse (Table 8) Type I 4-phosphatase. However, we did not identify an optimal phosphorylation sequence or an FXFP motif in this isoform. In contrast, we did not identify a D-domain in the human Type II 4-phosphatase (Table 7), although it does contain a consensus MAPK phosphorylation site. Human Type II 4-phosphatase also does not contain an FXFP motif.

The Sac phosphatase domain, a region of sequence homology found in several yeast, plant and animal proteins, can hydrolyze the 3-, 4-, or 5-position phosphate from PIs, although vicinal phosphate groups are resistant to hydrolysis 99100; thus, PtdIns3P, PtdIns4P and PtdIns(3,5)P₂ are substrates, whereas PtdIns(4,5)P₂ is not. The PtdIns4P phosphatase activity, but not PtdIns3P or PtdIns(3,5)P₂ phosphatase activities, of mammalian Sac1 complements phenotypic defects observed in yeast having deletions of Sac1p 101, indicating that the 4-phosphatase activity of these proteins is the most important in vivo. The human (Table 6) and mouse (Table 8) Sac1 proteins each contain five identifiable D-domains. Two of these D-domains are overlapping sequences (³⁴⁵KNMRWDRLSI and ³⁴⁸RWDRLSILL) and two are an alternative pair of D-domains (⁵¹⁷RDWKFLAL and ⁵¹⁷RDWKFLALPI). Neither the human nor mouse protein contains an optimal phosphorylation site for MAPKs or an FXFP motif.

The inositol polyphosphate 5-phosphatases are commonly classified on the basis of their substrate specificities 2750. In this system of classification, the Group I enzymes hydrolyze the water-soluble compounds Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄; the Group II enzymes hydrolyze both water-soluble and lipid substrates (e.g., PtdIns(4,5,)P₂ and PtdIns(3,4,5)P₃); the Group III enzymes hydrolyze the 3-phosphate-containing compounds, Ins(1,3,4,5)P₄ and PtdIns(3,4,5)P₃; and, the single Group IV enzyme hydrolyzes only the lipid substrates PtdIns(3,4,5)P₃ and PtdIns(4,5)P₂ 27102. Although we have used this classification system, we note that recent studies have demonstrated that the substrate specificities of several of the identified 5-phosphatases do not fit into this simple system 103104. For example, Schmid et al 103 have shown that there are significant differences in the substrate specificities of several "Type II" 5-phosphatases: synaptojannin 1, synaptojanin 2, the gene product responsible for Lowe's oculocerebrorenal syndrome (OCRL), skeletal muscle and kidney enriched phosphatase (SKIP), and INPP5B. Additionally, it should be noted that the in vitro and in vivo specificities of these enzymes may differ.

We did not identify a D-domain, FXFP motif or optimal MAPK phosphorylation sequence in either the human (Table 7) or mouse (Table 9) Type I 5-phosphatase. Several of the Group II 5-phosphatases (Tables 6 and 8) contain an identifiable D-domain. OCRL is the gene responsible for occulocerebrorenal dystrophy or Lowe's syndrome, when mutated 27. Both the human (Table 6) and mouse (Table 8) OCRL proteins contain a D-domain, but do not contain an FXFP motif or an optimal sequence for MAPK phosphorylation. Synaptojanin 1 and synaptojanin 2 are neuronal proteins that play a role in synaptic vesicle trafficking. They contain both a 5-phosphatase domain and a Sac phosphatase domain 99. The 5-phosphatase domain is responsible for the reported PtdIns(4,5)P₂-hydrolyzing activity of synaptojanins, while the Sac domain of synaptojanins accounts for their ability to also hydrolyze other PIs, such as the PtdIns4P product generated by the action of its 5-phosphatase domain 105. Both synaptojanin 1 and 2 contain optimal phosphorylation sites for MAPKs. However, only synaptojanin 2 contains a D-domain, which is conserved in the human (Table 6) and mouse (Table 8) proteins. The synaptojanin sequences that we searched do not contain an FXFP motif. The human (Table 6) and mouse (Table 8) 75-kDa inositol polyphosphate 5-phosphatases (inositol polyphosphate 5-phosphatase B) contain two conserved D-domains, but are devoid of an FXFP motif and an optimal sequence for MAPK phosphorylation.

The Group III SH2-containing inositol 5'-phosphatase 1 (SHIP1) is a hematopoietic-specific enzyme that hydrolyzes both PtdIns(3,4,5)₃ and Ins(1,3,4,5)P₄ 106. In addition, SHIP1 is able to hydrolyze the 4-phosphate from PtdIns(4,5) in vitro, thereby generating PtdIns5P 107. The human (Table 6) and mouse (Table 8) SHIP1 contain an identifiable D-domain. The human SHIP1 contains four optimal MAPK phosphorylation sequences; corresponding sequences for two of these are present in the mouse protein, whereas two are unique to the human protein. SHIP1 does not contain a sequence conforming to an FXFP motif. The distribution of SHIP2 is more ubiquitous than is that of SHIP1 106. The human SHIP2 is devoid of a D-domain or an FXFP motif (Table 7). It does contain four sequences that fit the optimal phosphorylation sequence for a MAPK. The human (Table 6) and mouse (Table 8) Group IV 5-phosphatase contain a conserved optimal sequence (PRSP) for MAPK phosphorylation; the human protein contains an additional optimal sequence of MAPK phosphorylation (⁵⁵PATP). Both the human and mouse Group IV 5-phosphatase contain an alternative pair of D-domains, as well as two other D-domains; neither contains an FXFP motif.

We examined each of the D-domains that we identified to determine if it overlaps with a KIM 14 or fits the MAPK-docking site consensus sequence defined by Bardwell and colleagues 1516. We found only one instance of overlap with a KIM: in the human PI 4-kinase α, the sequence ¹²LDERRRDAVIALGI not only contains the alternative pair of D-domains that we identified (Table 1), but also contains a KIM. Examination of the sequences found in Tables 1, 3, 6, and 8 revealed that, in several instances, the D-domain sequence fits the MAPK-docking sequence of Bardwell and colleagues. It should be noted that in several proteins we identified, but did not catalog, one or more sequence that conformed to the MAPK-docking motif of Bardwell and colleagues but did not conform to the more restrictive sequence that we used for a D-domain.

Finally, we also analyzed the sequences of PtdIns synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase) isozymes, which catalyze the production of PtdIns from cytidine diphosphodiacylglycerol and myo-inositol. We did not identify either an FXFP or a D-domain sequence in human or mouse PtdIns synthase sequences (GenBank:NP_006310, NP_665695, and NP_620093; data not shown). Further, we identified a MAPK phosphorylation site only in the human isoform 2. These results indicate that the isozymes that catalyze PtdIns synthesis are not likely to be directly regulated by MAPK; however, they do not rule out the possibility that MAPKs may control PtdIns synthesis via an effect on myo-inositol (e.g., via an effect on inositol monophosphatase A1) or cytidine diphosphodiacylglycerol levels.