The Interaction of Protein Kinase C Isozymes α, ι, and θ with the Cytoplasmic Domain of L-selectin Is Modulated by Phosphorylation of the Receptor

The leukocyte adhesion molecule L-selectin has an important role in the initial steps of leukocyte extravasation during inflammation and lymphocyte homing. Its cytoplasmic domain is involved in signal transduction after L-selectin cross-linking and in the regulation of receptor binding activity in response to intracellular signals. However, the signaling events occurring at the level of the receptor are largely unknown. This study therefore addressed the question of whether protein kinases associate with the cytoplasmic domain of the receptor and mediate its phosphorylation. Using a glutathione S-transferase fusion protein of the L-selectin cytoplasmic domain, we isolated a kinase activity from cellular extracts of the human leukemic Jurkat T-cell line that phosphorylated L-selectin on serine residues. This kinase showed characteristics of the protein kinase C (PKC) family. Moreover, the Ca2+-independent PKC isozymes θ and ι were found associated with the cytoplasmic domain of L-selectin. Pseudosubstrate inhibitors of these isozymes abolished phosphorylation of the cytoplasmic domain, demonstrating that these kinases are responsible for the phosphorylation. Analysis of proteins specifically bound to the phosphorylated cytoplasmic tail of L-selectin revealed that PKCα and -θ are strongly associated with the phosphorylated cytoplasmic domain of L-selectin. Binding of these isozymes to L-selectin was also found in intact cells after phorbol ester treatment inducing serine phosphorylation of the receptor. Furthermore, stimulation of Jurkat T-cells by CD3 cross-linking induced association of PKCα and -θ with L-selectin, indicating a role of these kinases in the regulation of L-selectin through the T-cell receptor complex. The phosphorylation-regulated association of PKC isozymes with the cytoplasmic domain of L-selectin indicates an important role of this kinase family in L-selectin signal transduction.

Leukocytes travel with the blood and lymph circulation throughout the body but have to leave the vessels to exert their immunological functions. Granulocytes migrate toward inflammatory sites in tissues (1), whereas lymphocytes home to secondary lymphoid tissues, where they become activated (2). These extravasation events are mediated by the orchestrated interaction of several adhesion molecule families that constitute an adhesion cascade, leading to recruitment of leukocytes to the vessel wall and migration through the endothelial cell layer (3,4).
L-selectin, a member of the carbohydrate binding selectin family of adhesion molecules, has an essential role in the initial steps of this adhesion cascade, the capture of leukocytes from the flowing blood ("tethering"), and the subsequent rolling along the vascular endothelium (5). L-selectin is a type I transmembrane protein with a lectin domain responsible for ligand binding, an epidermal growth factor domain, two short consensus repeats, a membrane-spanning region, and a short 17amino acid cytoplasmic domain without any conserved signaling motifs or interaction sequences (6). The cytoplasmic domain of the receptor is essential for L-selectin function because deletion mutants lacking the C-terminal 11 amino acid residues cannot support leukocyte rolling and binding of lymphocytes to high endothelial venules, although recognition of soluble ligand is not impaired (7). This defect has been attributed to a missing connection of the truncated L-selectin with the actin cytoskeleton, which might be mediated by the cytoskeletal linker ␣-actinin in intact L-selectin (8) and normally is induced by receptor ligation (9).
In addition to its adhesive function, L-selectin also mediates signaling in leukocytes. Ligation of L-selectin with antibody or ligand elicits of a number of cellular events, including elevated Ca 2ϩ and phosphotyrosine protein levels (10,11), synthesis of reactive oxygen compounds (12), activation of the mitogenactivated protein kinases extracellular-regulated kinase and Jun N-terminal kinase (13,14), and Rac-mediated rearrangements of the actin cytoskeleton (15). Furthermore, L-selectin triggers the enhanced binding activity of both ␤ 1 and ␤ 2 integrins (16 -19). Apart from transducing signals received from the outside of the cell, L-selectin itself is subject to regulation by cellular signals. Treatment of leukocytes with lineage-specific stimuli (cross-linking of CD2 or CD3 for lymphocytes, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor ␣ for neutrophils) leads to an increased binding of L-selectin to soluble ligand and of lymphocytes to high endothelial venule frozen sections (20). This enhancement of binding activity is assumed to be a result of phosphorylation of the cytoplasmic domain of the receptor on serine residues, which occurs constitutively at low levels and is significantly increased by stimulation with phorbol ester or chemokines through co-transfected G-protein-coupled chemokine receptors (21).
Although it is well established that signaling from and toward L-selectin occurs, the events at the level of the receptor are poorly understood. This study, therefore, aimed at the identification of cytosolic interaction partners (especially ki-nases) for L-selectin that are involved in receptor signaling.
Here we report that in T-cell lysates two PKC 1 isozymes, novel PKC and atypical PKC, phosphorylate the cytoplasmic domain of L-selectin on serine residues. Serine-phosphorylation of L-selectin increased association with PKC and induced binding of the conventional PKC␣ to the cytoplasmic tail of L-selectin in cell lysates and intact cells after activation with PMA. Moreover, lymphocyte activation through CD3 of the T-cell receptor complex induced binding of these PKC isozymes to L-selectin. These findings indicate a central role for PKC isozymes in the signal transduction events at the cytoplasmic domain of the L-selectin adhesion receptor.

MATERIALS AND METHODS
Reagents-All chemicals were obtained from Sigma or Merck unless stated otherwise. Purified histone H1 was from Calbiochem, myelin basic protein was from Invitrogen. Protein kinase inhibitors staurosporine and genistein were purchased from Alexis (Gruenberg, Germany), bisindolylmaleimide was from Calbiochem, and PKC and -pseudosubstrate peptide inhibitors were obtained from Biosource (Nivelles, Belgium). Hybridoma cell lines producing the mAbs Dreg200 and Dreg55 were kind gifts from Dr. E. C. Butcher, Stanford University. Expression and purification of mAb were described before (22). Isotype-specific PKC antibodies were purchased from BD Biosciences/Transduction Laboratories (Heidelberg, Germany).
Cloning of Glutathione S-Transferase (GST) Fusion Constructs-The sequence encoding the cytoplasmic domain of L-selectin was amplified by PCR using the full-length L-selectin cDNA in pCR3.1 (22) as a template. BamHI and EcoRI restriction sites were introduced by the PCR primers, and the fragment was subcloned in pGEX4T1 (Amersham Biosciences). A vector-encoded serine residue in the multiple cloning site, which could serve as a possible target for serine kinases with basic recognition sites, was changed to alanine by site-directed mutagenesis (QuikChange mutagenesis kit, Stratagene, Amsterdam, The Netherlands). Inactivating mutations of putative phosphorylation sites in the cytoplasmic domain were introduced by use of altered PCR primers. The resulting fragments were subcloned into pGEX4T1 as described for the wild type sequence.
Expression and Purification of GST Fusion Proteins-Escherichia coli strain BL-21 Codon Plus-RIL (Stratagene) transformed with pGEX4T1 or the fusion protein expression constructs were induced at mid-logarithmic growth phase with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside for 2 h at 37°C. Cells were harvested in buffer A (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1ϫ Complete® protease inhibitor mixture (Roche Applied Science)), containing 100 g/ml lysozyme and 0.1% Triton X-100 and disrupted by sonication. Lysates were cleared by centrifugation at 100,000 ϫ g, and supernatants were applied to a GSH-Sepharose column (Amersham Biosciences). After extensive washing with buffer A, fusion protein was eluted with 20 mM glutathione in 50 mM Tris, pH 8.0. Purity of isolated proteins was judged by SDS-PAGE and Coomassie staining, and concentration of purified protein was determined using the BCA assay (Pierce).
Isolation of Cellular Proteins Interacting with GST Fusion Protein ("Pull Down")-Jurkat T-cells were lysed in 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Brij 58, 1ϫ Complete® protease inhibitor mixture, 10 mM NaF, 1 mM sodium orthovanadate, and 100 g/ml RNase A on ice for 15 min, and lysates were cleared by centrifugation at 20,000 ϫ g for 20 min. Cell lysates were incubated with 10 l of GSH-Sepharose coated with GST or GST fusion protein. After 2 h beads were collected by centrifugation, washed 4 times with lysis buffer, and used in in vitro kinase assays, or bound proteins were eluted by boiling in SDS-sample buffer and analyzed by SDS-PAGE.
In Vitro Kinase Assay-To test for protein kinases in protein com-plexes bound to GST fusion proteins, isolated complexes were washed 3 more times with kinase buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MnCl 2 , 5 mM MgCl 2 ) and incubated in 50 l of kinase buffer containing 5 Ci of [␥-32 P]ATP (Amersham Biosciences) for 20 min at 30°C. Reactions were terminated by the addition of 450 l of phosphorylation stop buffer (10 mM sodium phosphate buffer, pH 8.0, 10 mM sodium pyrophosphate, 10 mM EDTA), and beads were washed 4 times with phosphorylation stop buffer containing 0.1% Triton X-100. For inhibitor studies protein complexes were preincubated with the indicated concentrations of inhibitor for 15 min before the addition of [␥-32 P]ATP. After the final washing step, protein complexes were resuspended in SDS sample buffer and subjected to SDS-PAGE. Proteins were visualized by staining with Coomassie Blue, and dried gels were analyzed by autoradiography. In experiments where additional substrates were used in the phosphorylation reaction, SDS sample buffer was added without further washing steps.
Immunoprecipitation-After treatment with 50 ng/ml PMA, 3 g/ml anti CD3 (UCHT1), or isotype-matched control antibody (both from BD Biosciences/Pharmingen) for 3 min at 37°C, Jurkat T-cells were lysed as described above with the exception that the lysis buffer contained 1% Nonidet P-40 as detergent. Cleared lysates were incubated for 2 h with anti-L-selectin mAb (a mixture of Dreg200 and Dreg55) coupled to sheep-anti-mouse-Ig-coated magnetic beads (Dynal, Hamburg, Germany). Immunoprecipitates were washed four times with lysis buffer, eluted by boiling in SDS sample buffer, and analyzed by SDS-PAGE, Western blotting, and immunodetection with isozyme-specific PKC mAb (BD Biosciences/Transduction Laboratories).
Mass Spectrometric Analysis of Proteins-For identification of PKC␣, bands were excised from Coomassie-stained SDS-polyacrylamide gels and in-gel digested with trypsin. The resulting peptide mixture was desalted using ZipTips (Millipore Corp) and analyzed by nanoelectrospray mass spectrometry. Mass spectra were acquired on a hybrid quadrupole time-of-flight mass spectrometer (Q-Tof, Micromass, Manchester, UK). The peptide sequence tag method (23) and de novo sequencing were used to identify the protein.
PKC tryptic peptides were analyzed by MALDI-TOF mass spectrometry using a Voyager-DE STR (Perseptive Biosystems, Framingham, MA). The peptide mass fingerprints obtained were analyzed searching the NCBI non-redundant protein and Swiss-Prot databases with the ProFound and Mascot software.  1B). The GST-LScyto fusion proteins as well as GST alone were expressed in E. coli BL21 and purified by glutathione (GSH) affinity chromatography from bacterial lysates. Purity of the eluted proteins was judged by Coomassie staining of SDSpolyacrylamide gels (Fig. 1C).

Expression of the Cytoplasmic Domain of L-selectin as a GST Fusion Protein in E. coli-For
The L-selectin Cytoplasmic Domain Associates with a Serine/Threonine Kinase Activity from Jurkat Cell Lysates-To isolate cellular kinases that are able to associate with and phosphorylate the cytoplasmic domain of L-selectin, GSH-Sepharose-coupled fusion protein GST-Lscyto was incubated with Jurkat T-cell lysates to allow binding of interacting proteins. The extensively washed protein complexes were then subjected to in vitro kinase assays to test for associated ki-nases. This assay revealed the presence of a kinase activity in the GST-LScyto-precipitated complexes that phosphorylated the fusion protein ( Fig. 2A, right lane). The GST part alone showed no incorporation of radioactivity ( Fig. 2A, left lane). A 60-kDa band of unknown origin was observed with GST as well as with GST-LScyto. Thus, to exclude that the kinase that phosphorylates GST-LScyto is actually bound to the GST portion of the fusion protein, we added GST-LScyto to GST isolates as an additional substrate during kinase assays. No phosphorylation of the cytoplasmic domain of L-selectin was observed in this experiment (Fig. 2B, right lane). Vice versa, the addition of GST to GST-LScyto precipitates did result in phosphorylation of GST-LScyto but not GST, showing that phosphorylation of the fusion protein does not occur on residues present in the GST part of the protein (Fig. 2B, left lane). Therefore, both binding of and phosphorylation by the associated kinase activity takes place at the L-selectin portion of the fusion protein.
Because the intracellular L-selectin sequence contains both serine and tyrosine residues which could serve as phosphoryl acceptor sites, we next aimed to characterize the specificity of the associated kinase activity. This kinase was able to strongly phosphorylate the classical serine/threonine kinase substrates histone H1 and myelin basic protein (MBP) (Fig. 3A). These findings were supported by the ability of the broad range serine/threonine kinase inhibitor staurosporine to abolish LScyto phosphorylation completely (Fig. 3B), whereas genistein, which blocks tyrosine kinases, had no effect on the activity of the L-selectin kinase.
Furthermore, replacement of both serines in the cytoplasmic sequence resulted in a complete loss of LScyto phosphorylation (Fig. 3C), whereas the kinase phosphorylated the fusion protein that carried an inactivated tyrosine to an extent comparable with the wild type protein. This demonstrated that the LScyto phosphorylation occurred exclusively on serine residues. Single serine-to-alanine replacements of the two potential phosphorylation sites revealed a preference of the kinase for serine 364 over serine 367, since inactivation of serine 364 resulted in a much stronger decrease in LScyto phosphorylation. Taken together, these results show that the cytoplasmic domain of L-selectin associates with, and is phosphorylated by, a serine kinase activity from T-cell lysates.
L-selectin Is an in Vitro Substrate for PKC and cGMP-dependent Protein Kinase -The serine residues in the cytoplasmic domain of L-selectin that are putative phosphoryl acceptor sites are surrounded by a high density of positively charged residues (Fig. 1A). To examine serine/threonine kinases that target basic sequences for the ability to use L-selectin as a substrate, in vitro phosphorylation assays with purified kinases were performed. Whereas protein kinase A showed very little phosphorylation activity toward GST-LScyto (data not shown), both PKC-and cGMP-dependent protein kinase strongly phosphorylated the fusion protein (Fig. 4). Further tests using protein variants carrying inactivating mutations of serine residues in the L-selectin sequence revealed that the two kinases can utilize both serines as substrates, although with different preferences. Whereas cGMP-dependent protein kinase-mediated phosphorylation was strongly impaired by the lack of serine 367 (Fig. 4A), phosphorylation by PKC, as observed for the cellular LScyto kinase, was more strongly affected by inactivation of serine 364 (Fig. 4B). The LScyto Kinase Is Stimulated by PKC Activators and Blocked by PKC Inhibitors-Because purified PKC and the LScyto-associating kinase activity from T-cell lysates displayed a similar phosphorylation pattern of the fusion protein, the effects of PKC-activating and -blocking substances on the phosphorylation of LScyto were further investigated. When cells were treated before lysis with the phorbol ester PMA, which can activate classical and novel PKC isozymes directly, the cellular GST-LScyto kinase activity was strongly stimulated (Fig. 5A). The PKC inhibitor bisindolylmaleimide I effectively blocked the phosphorylation of LScyto in a dose-dependent manner and at concentrations that are considered specific for this kinase (Fig. 5B). These results strongly indicated that members of the PKC family of kinases are responsible for the observed phosphorylation of the cytoplasmic domain of L-selectin.
The L-selectin Cytoplasmic Domain Associates with PKC in an Isozyme-specific Manner-Because our results pointed toward an involvement of PKC in the phosphorylation of Lselectin, we next examined whether PKC isozymes associate with the L-selectin cytoplasmic domain. To do so, GST-LScyto was incubated with Jurkat T-cell extracts, and bound protein complexes were tested for the presence of PKC by Western blotting using isozyme-specific monoclonal antibodies. Of the isozymes expressed in Jurkat cells, exclusively novel PKC and atypical PKC were detected in GST-LScyto pull down samples but not in GST controls. (Fig. 6, A and B). No association of the FIG. 3. The LScyto kinase activity is directed against serine residues. A, after isolation of LScyto-interacting protein complexes, 1 g of the indicated proteins were added to the kinase reaction to allow phosphorylation. H1, histone H1; MBP, myelin basic protein; G, GST; LS, GST-LScyto fusion protein; LScyto*, phosphorylated GST-LScyto. B, LScyto-associated proteins were incubated with 50 nm staurosporine (staur.) or 30 M genistein (geni.) for 20 min before the phosphorylation assay. C, the designated fusion proteins were used for precipitation of interacting proteins, and complexes were subjected to phosphorylation assays as described before. Upper panels, autoradiogram; lower panels, Coomassie-stained gel.

FIG. 4.
In vitro kinase assays with purified serine/threonine kinases. The indicated fusion proteins were used as in vitro substrates for purified kinases as described under "Experimental Procedures." A, in vitro kinase assay with cGMP-dependent protein kinase purified from bovine lung. B, in vitro kinase assay with PKC purified from rat brain. Upper panels, autoradiogram; lower panels, Coomassie-stained gel.
PKC isozymes ␣, ␤, ␥, ␦, ⑀, , or with GST-LScyto was found in this assays (data not shown). To test whether the two PKC isozymes associated with the cytoplasmic domain of L-selectin are responsible for the observed phosphorylation of this protein on serine residues, peptides that correspond to the intrinsic pseudosubstrate sequence (24) of the kinases were employed in inhibition assays. These sequences differ between members of the PKC family and allow isozyme-specific inhibition, which is difficult to achieve with chemical PKC inhibitors (25). LScyto pull down precipitates were incubated with PKC orpseudosubstrate peptides before the addition of [␥-32 P]ATP. Treatment with each one of the two pseudosubstrate inhibitors reduced phosphorylation of the fusion protein in a dose-dependent manner (Fig. 6C), indicating that both PKC isozymes are involved in L-selectin phosphorylation. Indeed, the extent of inhibition was increased when both pseudosubstrates were used simultaneously. These results clearly show that although various PKC isozymes could phosphorylate LScyto to a similar extent in vitro (data not shown), PKC and -shown to be associated with the cytoplasmic domain of L-selectin by immunoblotting (Fig. 6, A and B) indeed represent the cellular kinase activity that phosphorylates L-selectin (Fig. 2).
Identification of Phosphorylation-specific Interacting Proteins-Because phosphorylation of proteins involved in signal transduction frequently modulates their interaction capabilities with other signaling molecules, we examined whether the cytoplasmic domain of L-selectin associates with cytoplasmic proteins in a phosphorylation-dependent manner. For this purpose, LScyto was serine-phosphorylated by purified rat brain PKC, and interacting proteins were isolated from Jurkat T-cell lysates as described above. Analysis of bound protein complexes on silver-stained SDS-polyacrylamide gels revealed the presence of two bands of ϳ79 and 81 kDa that were only observed in association with phosphorylated LScyto but not with the unmodified cytoplasmic domain or GST controls (Fig.  7A). These protein bands were excised from Coomassie-stained SDS-polyacrylamide gels and analyzed by mass spectrometry. Both proteins were identified as PKC isozymes (Table I), p79 as novel PKC and p81 as conventional PKC␣. The presence of both proteins in the pull down eluates was confirmed in Western blots using isozyme-specific PKC antibodies (Fig. 7B). Because PKC␣ was also present in the rat brain PKC preparation used for phosphorylation of the fusion protein (Fig. 7B, upper  panel), it had to be excluded that the enzyme bound to GST-LScyto represents a contamination from the phosphorylation reaction. The initial indication that PKC␣ was derived from the cell lysate came from the observation that GST-LScyto, which had been phosphorylated by rat brain PKC but subsequently incubated with lysis buffer rather than Jurkat cell lysates, was not bound to detectable amounts of PKC␣ when analyzed in silver gels (Fig. 7A, fourth lane) or by more sensitive immunoblotting (Fig. 7B, upper panel, fifth lane). This was confirmed by the results of the tandem mass spectrometry analysis of the N-terminal peptide of the isolated protein. The sequence of this peptide corresponded to the sequence of the human rather than the rat isozyme, which differ in two residues. PKC␣ was solely associated with the phosphorylated LScyto fusion protein, and no binding was detectable with the non-phosphorylated cytoplasmic domain.
The PKC isozyme bound to phosphorylated GST-LScyto also originated from the Jurkat cell lysate, since this kinase was not detectable in the rat brain PKC preparation used for phosphorylation of the fusion protein (Fig. 7B, lower panel). In this experiment the previously shown association of PKC with the non-phosphorylated cytoplasmic domain of L-selectin was not detectable, since the amount of PKC that binds to the phosphorylated LScyto fusion protein (Fig. 7B) is significantly higher compared with the interaction with the non-phosphorylated protein shown in Fig. 6A. PKC bound to unphosphorylated LScyto was only detectable in highly concentrated eluates of the associating protein complexes but not visible in more diluted samples as used in Fig. 7. Phosphorylation of the cytoplasmic domain of L-selectin, thus, increases binding of PKC and induces association of PKC␣.
L-selectin Associates with PKC in Intact Cells-L-selectin has been reported to become phosphorylated on serine residues in various cell lines including Jurkat T-cells after stimulation with the phorbol ester PMA (21). To examine whether the PKC family members identified as interaction partners of the phosphorylated cytoplasmic domain of L-selectin in vitro also bind to the serine-phosphorylated receptor in intact cells, Jurkat T-cells were treated with PMA, and L-selectin was immuno- precipitated with antibodies directed against the extracellular part of the receptor. The precipitates were then tested for the presence of PKC isozymes. Both PKC␣ and -were detected in L-selectin immunoprecipitates from PMA-stimulated cells but not in precipitates from untreated cells, showing that this association in cells depends on serine phosphorylation of the receptor (Fig. 7C). The inability to detect the association of PKC and -with the non-phosphorylated L-selectin in vivo seen with the GST-LScyto fusion protein in vitro might be due to low amounts of associated kinase that are below the detection limit. Moreover, it appears likely that the cytoplasmic domain of L-selectin is occupied by alternative binding partners in resting cells, preventing constitutive interaction of the kinase with L-selectin. This experiment clearly demonstrates that isozyme-specific association of members of the PKC family with the cytoplasmic domain of L-selectin occurs in intact Tcells after serine phosphorylation of the receptor.

Stimulation of T-cells via the T-cell Receptor (TCR)
Induces PKC Binding to L-selectin-It has been shown previously that L-selectin binding activity in human blood lymphocytes is regulated via the CD3 receptor. L-selectin and CD3 are located in close proximity on the cell surface (40), and cross-linking of CD3 leads to increased L-selectin-dependent adhesion (20), which can be reversed by the serine/threonine kinase inhibitor staurosporine (21).
To verify the biological relevance of the interaction of PKC isozymes with the cytoplasmic tail of L-selectin, we examined the effect of CD3 cross-linking on the binding of PKC␣ and -to L-selectin. Stimulation of Jurkat cells with CD3 mAb induced association of both isozymes with L-selectin, whereas no binding was observed in non-activated cells or cells treated with isotype-matched control mAb (Fig. 8).
As shown in Fig. 7, binding of PKC␣ and -to the L-selectin tail is induced or up-regulated, respectively, by phosphorylation of the L-selectin cytoplasmic domain on serine residues. This phosphorylation dependence of the interaction was sup-ported by the observation that in intact cells, PKC␣ andbinding to L-selectin upon CD3 cross-linking is influenced by the specific PKC inhibitor bisindolylmaleimide. Although binding of PKC␣ was blocked completely by this kinase inhibitor, interaction with PKC was reduced but still detectable. This is in accordance with our observation that PKC can interact with the unphosphorylated cytoplasmic domain of L-selectin (Fig. 6A) and indicates that cellular stimulation allows increased access of the kinase to its binding site on L-selectin, which is not necessarily dependent on phosphorylation but rather on deactivation of alternative binding partners. The finding that PKC␣ and -become associated with the cytoplasmic domain of L-selectin after CD3 cross-linking supports the assumption that these two kinases are involved in the regulation of L-selectin binding activity through activation of the TCR complex.

DISCUSSION
In this study we report (i) that the cytoplasmic domain of L-selectin associates with a serine/threonine kinase activity from T-cell lysates that phosphorylates this sequence, (ii) that PKC and -are found in protein complexes that bind to the cytoplasmic domain and are responsible for the observed phosphorylation, (iii) that serine phosphorylation of the cytoplasmic domain strongly increases binding of PKC and induces interaction with PKC␣ in vitro and in intact cells, and (iv) that stimulation of T-cells via the T-cell receptor (CD3) complex induces binding of PKC␣ and -to L-selectin.
The PKC family of serine/threonine kinases consists of 10 members in human cells, which are under complex regulation by phosphorylation events, Ca 2ϩ concentration, and lipid cofactors (26,27). The PKC isozymes can be subclassified according to their domain structure and dependence on co-factors (28). The conventional or cPKCs (␣, ␤ I , ␤ II, ␥) are activated by diacylglycerol, the product of phospholipid cleavage by phospholipases. These isozymes also bind Ca 2ϩ , which is necessary for full activation by acidic membrane lipids. The novel or nPKCs (⑀, , ␦, ) are insensitive to Ca 2ϩ but still require diacylglycerol for activity. The regulatory mechanisms of the atypical or aPKCs and are not very well understood because they do not respond to classical PKC activators but in some cases show dependence on phosphoinositides and unsaturated fatty acids (29). Although the expression patterns of the individual isozymes are largely overlapping and the kinases show little substrate discrimination in vitro among the different members, research in recent years has shown distinct cellular functions for the PKC family members (30,31). This is achieved by coupling of the enzymes to different upstream-activating pathways and by selective subcellular distribution of the different isozymes. Although a range of isozymes from different subfamilies could phosphorylate LScyto to a similar extent (data not shown), our results show that interaction of L-selectin with PKC occurs in a strictly isozyme-specific manner. The cytoplasmic tail in its non-phosphorylated state interacts exclusively with novel PKC and atypical PKC but not with any other isozyme found in the Jurkat T lymphocyte cell line used to isolate L-selectin kinases (Fig. 6).
Phosphorylation of the LScyto fusion protein by these kinases TABLE I Mass spectrometric identification of proteins associating with the phosphorylated LScyto fusion protein p81 was subjected to tandem mass spectrometry electrospray ionization analysis. The amino acid sequences of the peptides given were determined with the sequence tag method (23) and by de novo sequencing. Residues differing between rat and human PKC␣ are underlined. p79 was analyzed by MALDI-TOF and peptide mass fingerprinting. B, LScyto-bound proteins were isolated as described above, blotted onto polyvinylidene difluoride membranes, and analyzed by immunodetection with mAb directed against PKC␣ and PKC together with whole cell lysate (WCL) and purified rat brain PKC. C, Jurkat T-cells were treated with 50 ng/ml PMA for 3 min and lysed, and L-selectin was immunoprecipitated with Dreg200 mAb. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with PKC␣ and PKC mAb.
was increased after stimulation of cells with phorbol ester and blocked by staurosporine. The same properties were previously described for the kinases that are responsible for the phosphorylation of L-selectin on serine residues in intact cells after chemoattractant receptor activation (21). It is, therefore, likely that PKC and -indeed represent the cellular L-selectin kinases.
The finding that the cytoplasmic tail of L-selectin associates with members of the PKC family corresponds well with previous reports that the cytoplasmic tail of L-selectin binds calmodulin in resting leukocytes (32). Interestingly, many calmodulinbinding proteins are also substrates for PKC. PKC and calmodulin usually compete for overlapping binding sites in these target proteins. Binding of calmodulin blocks access for the kinase, and phosphorylation of the sequence in return inhibits association with calmodulin (33). It is, therefore, conceivable that L-selectin, too, is regulated by such a mechanism. Dissociation of calmodulin from the cytoplasmic domain of Lselectin by calmodulin inhibitors or mutational inactivation of the binding site was shown to induce shedding of the receptor (32,34). This has been attributed to a conformational change in L-selectin, rendering the protein accessible for membrane-associated shedding proteases. In a cellular context, signaling events that induce dissociation of calmodulin from L-selectin might coincide with signals that activate PKC. This would lead to an immediate phosphorylation of the cytoplasmic domain after calmodulin dissociation and might delay shedding until PKC activity ceases. Phosphorylation of L-selectin on serine residues might allow further association with the cytoskeleton and other interaction partners that mediate the increased binding activity of the adhesion receptor.
In this context it is interesting that isolation of phosphorylation-specific interaction partners for the cytoplasmic domain revealed that phosphorylation of this sequence on serine residues strongly increased association of PKC and induced a comparably firm binding of the conventional PKC␣ not found with the non-phosphorylated cytoplasmic domain. The interaction was observed in vitro with phosphorylated LScyto fusion protein as well as in intact cells after PMA stimulation, inducing serine phosphorylation of L-selectin as reported previously (21). The observed strong association of phosphorylated L-selectin with PKC and -␣ exceeds transient kinase substrate interactions and is reminiscent of the interaction of PKC enzymes with proteins that mediate its subcellular distribution. Isozyme-specific PKC regulation is achieved apart from selective upstream activation by localization of the kinases to discrete cellular sites. This constitutes a mechanism to position the different isozymes in proximity to their specific activating signaling molecules and their phosphorylation substrates. Subcellular confinement is achieved by selective interaction of the PKC regulatory domain with a large number of PKC-interact-ing proteins, which in some cases also serve as PKC substrates (35,36). The stable association of serine-phosphorylated Lselectin with PKC␣ and -suggests that the cytoplasmic domain of this receptor acts as a PKC-anchoring protein. Binding to the cytoplasmic tail of L-selectin might, therefore, position the PKC isozymes close to substrates for further phosphorylation events. This is of particular interest, since several proteins that have been implicated in L-selectin function are also regulated through phosphorylation by the L-selectin-associated PKC isozymes.
Moreover, the novel PKC, which interacts both with the non-phosphorylated and phosphorylated cytoplasmic domain of L-selectin, displays a restricted expression pattern and is mainly found in T-cells and skeletal muscle (37), which may explain cell type-specific differences in L-selectin signaling. In T lymphocytes this isozyme has an essential role in TCR signal transduction leading to interleukin-2 expression by activation of the activator protein-1 (AP-1) and NF-B transcription factors. It becomes co-localized with the TCR in the immunological synapse at the contact region between lymphocytes and antigen-presenting cells (38). PKC is recruited to the TCR complex by direct interaction with CD4-associated Lck-tyrosine kinase (39). Tyrosine phosphorylation by Lck at Tyr90 seems important for PKC activation because exchange of this residue to alanine resulted in the inability of the kinase to induce cell proliferation. Intact Lck has been shown to be involved in a number of L-selectin-induced signaling pathways in T-cells (13)(14)(15), but direct interaction between Lck and the cytoplasmic domain has not been reported. PKC could provide the link that couples L-selectin to the tyrosine kinase Lck and to further downstream signaling events. Furthermore, functional cross-talk between L-selectin and the T-cell receptor complex has been described. Immobilized antibodies directed against L-selectin provide a co-stimulatory signal for lymphocyte activation via the TCR (40). Vice versa, stimulation of T-cells via CD3 increases the binding activity of L-selectin (20). Because L-selectin and the TCR are found in close proximity on the cell surface and L-selectin co-precipitates with the CD3 chain, PKC or/and Lck could be the point of intersection between the signaling pathways of both receptors.
Moesin, a cytoskeletal linker of the ERM (Ezrin-Radixin-Moesin) family, has been shown to bind to peptides corresponding to the cytoplasmic domain of L-selectin (41). This interaction only occurs after stimulation of cells with PMA and involves a subpopulation of moesin which is phosphorylated at a C-terminal sequence that contains three threonine residues. The phosphorylation in this actin binding domain disrupts intramolecular association between the N and C termini and is thought to activate moesin. Thr-558, one of the threonine res- idues located in the actin binding region, was identified as a target for phosphorylation by PKC (42). Hence, receptorbound PKC could induce connection of L-selectin with the actin cytoskeleton by phosphorylation of local moesin pools and activation of its binding activity.
Co-localization of L-selectin has also been shown in the case of the CD18 chain of ␤ 2 integrins, which are activated by L-selectin cross-linking (18,19,43). Recently, it was shown that PKC␣ phosphorylates CD18 in a threonine-rich motif (44), which has been implicated to be involved in the up-regulation of integrin binding activity (45) and association with the actin cytoskeleton (46,47). Because PKC␣ associates with the cytoplasmic tail of L-selectin, this could constitute the mechanism to bring the active kinase into proximity to the CD18 chain, allowing phosphorylation and activation of the integrin.
Although the function of the PKC isozymes in L-selectin signaling remains to be examined in more detail, the data of this study and of previous reports (20,21) indicate that serine phosphorylation of L-selectin by PKC has physiological roles in the regulation of L-selectin adhesive activity. Stimulation of T-cells via the T-cell receptor complex by CD3 cross-linking has been shown to increase L-selectin-dependent cell adhesion to high endothelial venules (21). Correspondingly, CD3 crosslinking induced the phosphorylation-dependent binding of PKC␣ and stimulated binding of PKC to the cytoplasmic domain of L-selectin as demonstrated in the present study (Fig.  8). Staurosporine, which inhibits serine phosphorylation of Lselectin in vivo (21) as well as in vitro (Fig. 3B), blocked the increased binding of T lymphocytes to high endothelial venules after cross-linking of CD3 (21). Likewise, the PKC inhibitor bisindolylmaleimide inhibited PKC and -␣ association with L-selectin induced by CD3 cross-linking (Fig. 8). Regulation of the L-selectin adhesive activity through PKC isozymes could be achieved by controlling the anchoring of the receptor to the cytoskeleton or to other cytoplasmic ligands. Serine phosphorylation of the L-selectin cytoplasmic tail by PKC was shown to be induced by stimulation via chemokine receptors and G proteins (21). The association of L-selectin with PKCs that belong to differently regulated subclasses of this kinase family indicates that serine phosphorylation of the receptor is under the control of additional diverse upstream regulatory pathways.
In summary, the finding of this study that specific PKC isozymes associate with the cytoplasmic domain of L-selectin and mediate its phosphorylation, that binding of PKC␣ andto the phosphorylated cytoplasmic domain is induced by cell stimulation via the T-cell receptor (CD3) complex in conjunction with previous reports of chemoattractant receptor-induced L-selectin phosphorylation (21) clearly shows that PKC isozymes have an important role in L-selectin-related signal transduction events.