JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M210596200 on April 9, 2003

J. Biol. Chem., Vol. 278, Issue 26, 24072-24077, June 27, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/26/24072    most recent
M210596200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, X.
Right arrow Articles by Sun, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, X.
Right arrow Articles by Sun, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Functions of the Activation Loop in Csk Protein-tyrosine Kinase*

Xiaofeng Lin, Sungsoo Lee and Gongqin Sun {ddagger}

From the Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881

Received for publication, October 16, 2002 , and in revised form, February 21, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autophosphorylation in the activation loop is a common mechanism regulating the activities of protein-tyrosine kinases (PTKs). PTKs in the Csk family, Csk and Chk, are rare exceptions for lacking Tyr residues in this loop. We probed the function of this loop in Csk by extensive site-specific mutagenesis and kinetic studies using physiological and artificial substrates. These studies led to several surprising conclusions. First, specific residues in Csk activation loop had little discernable functions in phosphorylation of its physiological substrate Src, as Ala scanning and loop replacement mutations decreased Csk activity toward Src less than 40%. Second, some activation loop mutants, such as a single residue deletion or replacing all residues with Gly, exhibited 1–2% of wild type (wt) activity toward artificial substrates, but significantly higher activity toward Src. Third, introduction of a thrombin cleavage site to the activation loop also resulted in loss of 98% of wt activity for poly(E4Y) and loss of 95% of wt activity toward Src, but digestion with thrombin to cut the activation loop, resulted in full recovery of wt activity toward both substrates. This suggested that the catalytic machinery is fully functional without the activation loop, implying an inhibitory role by the activation loop as a regulatory structure. Fourth, Arg313, although universally conserved in protein kinases, and essential for the activity of other PTKs so far tested, is not important for Csk activity. These findings provide new perspectives for understanding autophosphorylation as a regulatory mechanism and imply key differences in Csk recognition of artificial and physiological substrates.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein tyrosine phosphorylation plays crucial roles in regulating cell proliferation and differentiation, the cell cycle, and transmission of various signals from the extracellular environment to a cell (1). This post-translational modification is catalyzed by protein-tyrosine kinases (PTKs),1 a large class of enzymes that transfer the {gamma}-phosphate of ATP to specific tyrosine residues in their protein substrates. Due to their critical roles in cellular regulation, the activities of PTKs in the cells are tightly controlled. Autophosphorylation is a key mechanism regulating the activity of most PTKs (2, 3). It occurs on one or multiple Tyr residues on a loop located in the kinase domain, called the autophosphorylation loop. Since autophosphorylation activates the kinases, this loop is also called the activation loop.

The catalytic domain of a PTK consists of an ATP-binding lobe, a peptide binding lobe, and a cleft between the two lobes (4). The cleft is the active site and contains two loops: the activation loop and the catalytic loop. The activation loop controls kinase activities by assuming different phosphorylation dependent conformations (3, 5). In the unphosphorylated form, the activation loop blocks the access to the active site by interactions with residues from the catalytic loop and other substructures within the ATP- and peptide-binding lobes. In this conformation the autophosphorylatable Tyr forms a hydrogen bond with a catalytically essential Asp residue located in the catalytic loop (4, 6, 7). Autophosphorylation results in large conformational changes in the activation loop that moves away from the active site, making the active site accessible (8, 9). The active conformation is stabilized by interactions of the phospho-Tyr in the activation loop with two Arg residues, one from the catalytic loop and the other from the activation loop (8, 9).

In light of the virtual ubiquity of autophosphorylation as a key regulatory mechanism, it is intriguing that PTKs in the Csk family, COOH-terminal Src kinase (Csk) and Csk homologous kinase (Chk), do not contain a Tyr residue in the activation loop (10, 11). Csk and Chk are soluble PTKs that phosphorylate PTKs of the Src family on a COOH-terminal Tyr and negatively regulate their activities (12). The kinase domains of Csk (Refs. 13 and 14 and Fig. 1A) and Chk share many structural features with other PTKs, such as the ATP-binding and peptide-binding lobes, and the active site cleft. Within the active site, Csk also contains the catalytic loop and the activation loop. Their catalytic loops share the consensus sequence of HRDLAARN with other PTKs. Like other PTKs, the Csk and Chk activation loops follows a Asp332-Phe-Gly (DFG) motif on the amino end and connect to p + 1 loop on the carboxyl end. But the Csk and Chk activation loops share little homology with other PTKs or with each other. Since Csk and Chk do not contain a Tyr residue in the activation loop, they appear not regulated by autophosphorylation (15, 16). It is not clear if this loop is involved in some alternative regulatory mechanisms.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1.
Csk structure and the conformations of the activation loop. A, crystal structure of full-length Csk showing the SH3 and SH2 domains, ATP-binding lobe (A-lobe), peptide-binding lobe (P-lobe), the catalytic and activation loops in the active site. B, conformations of the activation loop in active (a) and inactive (b) Csk. The residues that align, such as Phe333 and Thr336, are labeled together, and those that do not align, such as Asp332, Lys337, and Lys347, are labeled separately. Some residues in the activation loop were not resolved in the crystal structure and not shown. The alignment was performed by K2 protein structure alignment program (39).

 

In this communication, we probed the role of the activation loop of Csk by site-specific mutagenesis and kinetic characterization. Several observations on this exceptional activation loop provide novel insights into the role of activation loops, substrate recognition, and cooperation between the regulatory and catalytic machinery of the PTK structure.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Csk Mutants—(His)6-tagged Csk was expressed using pET-Csk-H6 (constructed by inserting human Csk coding sequence with a (His)6 tag sequence at the 3'-end into the pET-23 a(+)) in BL21 (DE3) Escherichia coli. The full-length Csk was extended by 6 His residues at the COOH terminus to aid purification. Ala scanning mutants and other Csk point mutants were generated using QuikChange (Strategene) in the parental plasmid of pET-Csk-H6. Loop replacement mutation (CSAL) was incorporated by polymerase chain reaction with primer extensions. All mutants were confirmed by DNA sequencing.

Enzyme Purification—The wt and mutant Csk enzymes were purified to apparent or near homogeneity by Ni2+-affinity chromatography as follows. Harvested cell paste was resuspended (culture volume versus resuspension volume = 50:1) in the binding buffer (100 mM Tris-Cl and 5 mM imidazole, pH 8.0) and sonicated three times for 10 s each. The cell lysate was then cleared by centrifugation at 34,000 x g for 10 min. The supernatant was applied to a Ni2+-iminodiacetic acid-agarose column (Sigma) equilibrated with the binding buffer. After the sample was loaded, the column was washed with 15 bed volumes of the wash buffer (100 mM Tris-Cl and 60 mM imidazole, pH 8.0). The protein still bound to the column was eluted with elution buffer (100 mM Tris-Cl and 200 mM imidazole, pH 8.0). The purified enzymes were desalted on a Sephadex G25 column equilibrated with the storage buffer (100 mM Tris-Cl, pH 8.0, and 0.1% {beta}-mercaptoethanol). Glycerol was added to the purified fractions to 30%, and the enzymes were stored at –20 °C. Protein concentrations were determined by Bradford assay and purity was assessed by SDS-PAGE with Coomassie Blue staining.

Kinase Activity Assay—For assaying PTK activities, we measured the phosphorylation of polyE4Y and kdSrc using acid precipitation assay as described previously (17, 18). The kinase-defective Src (kdSrc) was avian Src that contained a Lys295Met mutation, which abolished its kinase activity (18, 19). When the Km and kcat were determined with regard to one substrate, kinase activity was determined at various concentrations of that substrate in the range of 20–200 µM for ATP, 20–200 µg ml–1 for poly(E4Y), and 1–10 µM for kdSrc. The kcat and Km values were determined by Lineweaver-Burk plots with linear regression using the Microsoft Excel program. Carboxymethylated-maleylated, reduced lysozyme (RCM-L) and GST fused to the Csk substrate peptide (GCSP) were also used as substrates in certain assays. GCSP was generated by fusing the optimal Csk peptide sequence, EEIYFFF (20), to the carboxyl terminus of GST.

Thrombin Digestion—Equal amounts (20 µg) of wt Csk, or the Csk mutant TC-Csk, were incubated in the presence or absence of 0.8 µg of thrombin (~2 NIH units) in the thrombin digestion buffer (50 mM Tris, pH 8.0) at 25 °C for 2 h. At the end of the digestion, aliquots of the protein samples were mixed with equal volumes of sample buffer and analyzed by SDS-PAGE. For enzymatic characterization of thrombin-digested TC-Csk, the digested enzymes were stored at 4 °C and used within the same day. Storage of the digestion mixture at 4 °C for up to 1 day did not result in detectable nonspecific digestion or loss of kinase activity.

To determine whether the two fragments produced by thrombin digestion were associated with each other, digested TC-Csk was purified by Ni2+-affinity column as described earlier. The purified proteins were compared with digested TC-Csk prior to this step of affinity purification to determine the co-purification of digested fragments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ala Scanning Mutagenesis of the Csk Activation Loop— A comparison between active and inactive structures of Csk (14) indicated that many residues in the activation loop assume dramatically different conformations (Fig. 1B) in the two forms, suggesting the possibility that the conformation of the activation loop may correlate to the activation states of Csk. To evaluate the role of individual residues in this loop, all non-Ala residues were individually mutated to Ala. The kinase activities of mutants were determined using both poly(E4Y) and the kdSrc as substrates. Among the 10 mutants, most exhibited specific activity within 80% of the wt enzyme (Fig. 2). The most severe effect was observed in Glu338 -> Ala, which retained ~40 and 60% of wt activity using poly(E4Y) and kdSrc as substrates, respectively. In contrast, mutation of Asp332 of the DFG motif in front of the activation loop to Ala abolished the kinase activity with either substrate. These results indicated that none of the mutated residues in the Csk activation loop played any essential roles in Csk catalysis or activation.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2.
Ala scanning mutagenesis of the Csk activation loop. A, sequence of the Csk activation loop. The variable region to be studied is not underlined. B, activity of Ala mutants using polyE4Y and Src as substrates. Asp332 -> Ala and Arg313 -> Ala are also included. S.E. values are shown. Invisible error bars indicate small errors to be seen in the graph.

 

In other PTKs that are regulated by autophosphorylation, the Tyr(P) in the activation loop is stabilized by interaction with two Arg residues. The first Arg, which is equivalent to Lys337 in Csk, is critical for the autophosphorylation-initiated activation of v-Fps (21), but our results demonstrated that Lys337 was not required for Csk activation. The second is an Arg from the catalytic loop, equivalent to Arg313, which is universally conserved. To determine whether Arg313 is required for Csk activation, we mutated Arg313 to Ala. Arg313 -> Ala exhibited 87 and 108% of the wt activity toward poly(E4Y) and kdSrc, respectively (Fig. 2). This result indicated that the side chain of Arg313 was not involved in essential catalytic or regulatory functions. This effect directly contrasts mutagenic studies of Src and v-FPS, in which mutations of the equivalent residues to Ala abolished the kinase activities (21, 22).

The Csk Activation Loop Can Be Functionally Replaced by an Unrelated Activation Loop—Csk shares the highest overall amino acid sequence homology with PTKs of the Src family, but the two families share no significant homology in the activation loop. Src contains one Tyr residue in the activation loop, whose autophosphorylation activates Src kinase activity, and alters other regulatory properties (23–25). The Src activation loop is also four residues longer than the Csk counterpart. To determine whether the Csk activation loop can be functionally replaced by the Src counterpart, a Csk mutant with a Src activation loop (CSAL) was generated. CSAL exhibited ~80 and 130% of the wt activity with polyE4Y and kdSrc as substrates, respectively, indicating that the longer activation loop can be accommodated and that none of the Csk-specific residues in the activation loop was required for the kinase activity. The Ala scanning and the loop replacement results indicate that Csk is active without the help of activation loop side chains.

We then determined whether CSAL, containing a Tyr residue in the activation loop, could autophosphorylate and whether the phosphorylation of the Tyr residue could activate CSAL (Fig. 3). Incubation of the mutant enzyme with 0.1 mM ATP and 12 mM Mg2+ did not result in autophosphorylation higher than that of wt Csk. When incubated with Src in the presence of ATP and MgCl2, CSAL, but not Csk, was readily phosphorylated in a time-dependent manner. This result indicated that the amino acid sequence in the activation loop contained the necessary structural information to be phosphorylated by Src, but not by Csk. The phosphorylation of CSAL by Src is also consistent with the trans-mechanism for Src autophosphorylation (26). No effect on CSAL activity was detected when the phosphorylation level reached ~30% (data not shown).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 3.
Autophosphorylation and trans-phosphorylation of CSAL. A, sequence comparison between Csk and Src activation loop. DFG motif, activation loop, and the p + 1 loop are indicated. The conserved residues are in bold. B, Csk and CSAL autophosphorylation and trans-phosphorylation by Src. Each lane contains 2 µg of Csk or CSAL. 1 ng of autophosphorylated Src is present in indicated lanes. C, time course of Csk and CSAL phosphorylation by Src. Each lane contains 2 µg of Csk or CSAL and 1 ng of autophosphorylated Src.

 

Probing the Conformational Role of the Csk Activation Loop—The apparent lack of a functional role by the activation loop contradicted our expectations based on its central location and the conformational differences between active and inactive Csk. We further probed the role of the activation loop by additional more aggressive mutations (Table I).


View this table:
[in this window]
[in a new window]
  		TABLE I
Kinetic parameters of wt and selected Csk mutants

Standard errors are presented. These values were calculated from three experiments, with each one performed in duplicate.

 

We determined whether the overall length of the loop was important by deleting Ala339 ({Delta}339). This mutant retained ~1.8% of the wt activity toward poly(E4Y) and 20% wt activity toward kdSrc. Since the Ala side chain was unlikely to be essential for catalysis or regulation, this result suggested that the length or the conformation of the activation loop was important for full activity, but apparently more so for poly(E4Y) than for Src. This result suggested that although the activation loop was not part of the catalytic machinery, its conformation could have a dramatic effect on the activity of Csk in a substrate dependent manner.

Since none of the side chains in the activation loop was essential, we determined if we could remove all side chains from this region and retain Csk activity. An all-Gly activation loop mutant (GAL), in which Gly replaced all 11 activation loop residues, was constructed. The mutant exhibited 1 and 14% of wt activity toward poly(E4Y) and Src, respectively (Table I). The differential phosphorylation of poly(E4Y) and Src by GAL was similar to {Delta}339 and further confirmed that the side chain functional groups were not required for catalysis, but the conformation of the loop affected the kinase activity in a substrate-dependent manner. Although the effects of the activation loop mutations were apparently substrate-dependent, only kcat values, but not the apparent Km values for the two substrates used, were significantly affected. This suggested that phosphoryl transfer onto these substrates, instead of the recognition of these substrates by Csk, was differentially affected by the activation loop mutations. The mutants displayed similar relative activity toward two other artificial substrates, RCM-lysozyme and GST fused to the optimal Csk substrate peptide, as toward poly(E4Y), suggesting that the mutants differentiated the physiological substrate from the artificial substrates.

Csk Is Fully Active without an Intact Activation Loop—Since the activation loop residues were not required for Csk function, we wanted to determine the effect of cleaving the activation loop. A mutant that contained a thrombin cleavage sequence in the activation loop (TC-Csk) was generated, in which the thrombin cleavage sequence of LVPRGS replaced ASSTQD in the activation loop. TC-Csk retained ~2% wt activity toward poly(E4Y) and ~5% wt activity toward kdSrc (Table I).

Fig. 4 shows the thrombin digestion on TC-Csk (Fig. 4A) and its effect on Csk activity (Fig. 4B). Incubation with buffer alone or thrombin did not cleave or affect the activity of wt Csk (data not shown). Incubation with buffer alone did not lead to cleavage of TC-Csk or significant change in its kinase activity compared with TC-Csk kept on ice. Incubation with thrombin led to cleavage of TC-Csk into two fragments at 38 and 12 kDa, respectively, the expected sizes when the thrombin cleavage site was digested. This cleavage activated TC-Csk, resulting in full recovery of the wt Csk activity toward both poly(E4Y) and kdSrc. This result indicated that the low activity of TC-Csk was due to a conformational effect, which could be released by thrombin cleavage, and that an intact activation loop was not required for full function of Csk.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4.
Digestion of TC-Csk by thrombin. 20 µg of Csk or TC-Csk were incubated with either buffer or 0.8 µg of thrombin (2 NIH units) for 2 h, and the digested proteins were analyzed by SDS-PAGE (A) or kinase activity assay using poly(E4Y) (closed bar) and kdSrc (open bar) as the substrates (B).

 

The two fragments produced by thrombin digestion were both essential parts of the kinase core structure (Fig. 5A). It was surprising that the two parts could still make up a fully active enzyme. To confirm that the two fragments were indeed associated with each other after thrombin digestion, the digested enzyme was purified on a Ni2+-IDA affinity column. Since only the 12-kDa fragment contained the poly(His)6 tag, co-purification of both fragments on a Ni2+-affinity column would indicate that the two fragments were associated with each other. SDS-PAGE indicated that the relative ratios of the two fragments were similar before and after Ni2+-affinity purification, thus confirming that the two fragments were strongly associated with each other (Fig. 5B). Examination of the crystal structure of Csk indicated that the two parts were held together by extensive hydrophobic interactions between the side chains on 3 {alpha}-helices: {alpha}E from the 38-kDa fragment and {alpha}F and {alpha}I on the 12-kDa fragments (Fig. 5A).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 5.
Hydrophobic interactions holding Csk fragments together. A, thrombin digestion in the activation loop cleaves Csk into 38-kDa (in red) and 12-kDa (in blue) fragments. Extensive hydrophobic interactions among the side chains from a helices E, F, and I likely hold the two fragments together. B, co-purification of the two fragments by (His)6 affinity chromatography. Lane 1, TC-Csk; lane 2, Digested TC-Csk; and lane 3, digested TC-Csk after (His)6 affinity chromatography.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
PTKs of the Csk family are unusual in that they lack an autophosphorylation site in the activation loop, a regulatory mechanism conserved among all other PTKs. Considering the central location of this loop in the active site and well demonstrated roles of autophosphorylation in the regulation of other PTKs, we probed the function of this loop in Csk with some surprising findings.

The Activation Loop of Csk Is Not Essential for the Kinase Function—None of the mutations, including Ala scanning, loop replacement, site-specific deletion, replacing all residues by Gly, and cleaving the activation loop, completely abolished Csk activity. The lowest activity toward kdSrc was observed for TC-Csk, at 5%, but digestion of the activation loop with thrombin restored the activity completely. Similar recovery of kinase activity toward poly(E4Y) was also observed, indicating that the low activity in TC-Csk, and likely in other mutants, was not due to the loss of any functionalities in the activation loop, but an inhibitory conformation. These results indicated that the activation loop is not part of the catalytic machinery of Csk, nor is it required for the catalytic machinery to be fully functional.

Another important question is whether conformational changes in the activation loop do serve as a regulatory element in Csk, and if so, what regulatory signals can bring about such conformational changes. The SH3 and SH2 domains of Csk interact with and increase the activity of the catalytic domain (27–29). As discussed earlier, the active and full-length Csk and inactive Csk lacking the regulatory SH2 and SH3 domains have major differences in the conformation of the activation loop (13, 14). It appears likely that interactions between the regulatory domains with the catalytic domain can affect the conformation of the activation loop. Whether conformational changes in the activation loop mediate the SH2-SH3 regulation of Csk awaits further investigation.

If the catalytic machinery between Csk and other PTKs are fundamentally the same, then these results suggested that the Csk activation loop was an evolutionally "basal" activation loop devoid of regulatory functionalities. The activation loops in other PTKs acquired other functionalities that allow them to interact with and regulate the function of the catalytic machinery. Based on this perspective, we propose the following functionalities for the activation loop in other PTKs. First, the amino acid sequence itself may interact with the catalytic machinery and act as an inhibitor. Second, the amino acid sequence surrounding the Tyr residue is needed to render the Tyr residue a good substrate for the kinase. Third, the phosphorylated activation loop needs to form certain interactions so the inhibitory interactions are removed. Finally, the catalytic machinery and the activation loop may evolve in a corresponding fashion, so the potential regulation by the activation loop can be accommodated. Mutagenic and structural studies of insulin receptor kinase, which is regulated by autophosphorylation, are consistent with these perspectives (30–32). These perspectives may provide a base line for dissecting autophosphorylation as a key mechanism for PTK regulation.

Consistent with this proposal, the catalytic machinery of Csk appears to be different from that of other PTKs. Arg313 is universally conserved among protein kinases (33), and the corresponding residue is essential in two PTKs that have been tested: mutations of the equivalent residue in Src (Arg385) (21) and v-Fps (Arg1042) (22) resulted in inactive (<0.1% of wt activity) enzymes. But mutation of Arg313 had no effect on the Csk activity toward either the physiological or artificial substrates. This suggests the possibility that the role of this Arg residue has co-evolved with that of the activation loop: in PTKs that autophosphorylate, this Arg needs to interact with the phospho-Tyr for the kinase to be active; in PTKs that lack autophosphorylation, the catalytic machinery does not require the activation mediated by this residue. In Ser protein kinases, the equivalent residue also plays an important although not essential role. Mutation of the corresponding Arg residue to Ala decreased the kinase activity by 90% in yeast cAMP-dependent protein kinase (Arg209) (34), and 80% in glycogen phosphorylase kinase (35, 36), indicating an important regulatory, but not a catalytically essential function for this residue.

Some Csk Activation Loop Mutants Differentially Phosphorylate the Physiological and Artificial Substrates—{Delta}339 and GAL exhibited only 1 and 2% of wt activity toward poly(E4Y) but 14 and 18% wt activity toward kdSrc, indicating that the effect of the activation loop mutations are substrate-dependent. These observations raise an important question about the mechanisms Csk substrate recognition. Theoretically, there are two ways alterations in the activation loop could be propagated to other parts of the enzyme to affect the kinase activity. The first is by direct interaction of the side chains in this region. But Ala scanning and loop replacement results eliminated the possibility that the side chains in the activation loop are directly involved in regulation or catalysis. The second way the alteration can be propagated is by affecting the conformation of fragments that are covalently linked to the activation loop. The amino end of the activation loop is connected to the conserved DFG motif and the carboxyl end of the activation loop is linked to the p + 1 loop. The DFG motif is catalytically essential as the mutation of Asp332 -> Ala abolished the kinase activity toward both polyE4Y and Src. The p + 1 loop is responsible for interacting with peptide substrates (9). Propagation of the conformational changes to either DFG or p + 1 loop would likely dramatically alter the kinase activity. Since the mutational effects were substrate-dependent, it suggested that the conformational changes in the activation loop predominantly affected the function of the p + 1 loop.

Then how do such conformational changes distinguish the artificial substrates from the physiological substrate? One explanation is that the phosphorylation of the artificial substrate predominantly relies on its interactions with the activation loop and p + 1 loop, while the phosphorylation of Src relies less on such interactions. This implies that Src also interacts with Csk in regions outside the activation loop/p + 1 loop. This interpretation is consistent with our understanding of peptide substrate binding to PTKs (9) in general and consistent with the complex Csk-Src interactions that has been suggested by two lines of evidence. 1) Although Tyr527 in Src is an excellent substrate for Csk (18, 19), peptides mimicking the tail sequence of Src (19, 37)2 or heat-inactivated Src (38) are much worse substrates compared with Src or to the optimal Csk peptide substrate, suggesting that Tyr527 recognition by Csk relies on more than just the local peptide sequence. 2) Mutations of the residues surrounding Tyr527 of Src have only relatively minor effect on the ability of Src to be phosphorylated by Csk (19). These observations indicate that some interactions that make Tyr527 of Src a good substrate for Csk are not located on the tail sequence surrounding the phosphorylation site, but in other regions of Src. Such a "bivalent Csk-Src" interaction model could provide a basis for the differential effects by the activation loop mutations. The nature and details of the Csk-Src interaction remain to be determined.


   FOOTNOTES
 
* This work was supported by Grant 1 P20 RR16457 from the National Institutes of Health and by a grant from the University of Rhode Island Research Council. DNA sequencing was performed at the M.D. Anderson Cancer Center Core Sequencing Facility (supported by National Institutes of Health Grant CA16672). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, 117 Morrill Science Bldg., 45 Lower College Rd., University of Rhode Island, Kingston, RI 02881. Tel.: 401-874-5937; Fax: 401-874-2202; E-mail: gsun{at}uri.edu.

1 The abbreviations used are: PTK, protein-tyrosine kinase; Chk, Csk-homologous kinase; CSAL, Csk with a Src activation loop; Csk, COOH-terminal Src kinase; {Delta}339, Csk mutant in which residue 339 is deleted; GAL, Csk with all activation loop residues replaced by Gly; GCSP, GST fused to Csk substrate peptide; GST, glutathione S-transferase; kdSrc, kinase-defective Src; RCM-L, carboxymethylated-maleylated, reduced lysozyme; TC-Csk, thrombin-cleavable Csk; wt, wild type. Back

2 X. Lin, S. Lee, and G. Sun, unpublished results. Back


   ACKNOWLEDGMENTS
 
Generous help from P. Lee at M.D. Anderson Cancer Center for the generation of the CSAL mutant is gratefully acknowledged. We thank P. Cohen and Marina Ayrapetov for critically reading the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hunter, T. (1998) Philos. Trans. R. Soc. Lond. B Biol. Sci. 353, 583–605[CrossRef][Medline] [Order article via Infotrieve]
  2. Smith, J. A., Francis, S. H., and Corbin, J. D. (1993) Mol. Cell. Biochem. 127–128, 51–70
  3. Hubbard, S. R., and Till, J. H. (2000) Annu. Rev. Biochem. 69, 373–398[CrossRef][Medline] [Order article via Infotrieve]
  4. Hubbard, S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994) Nature 372, 746–754[CrossRef][Medline] [Order article via Infotrieve]
  5. Huse, M., and Kuriyan, J. (2002) Cell 109, 275–282[CrossRef][Medline] [Order article via Infotrieve]
  6. Sicheri, F., Moarefi, I., and Kuriyan, J. (1997) Nature 385, 602–609[CrossRef][Medline] [Order article via Infotrieve]
  7. Xu, W., Harrison, S. C., and Eck, M. J. (1997) Nature 385, 595–602[CrossRef][Medline] [Order article via Infotrieve]
  8. Yamaguchi, H., and Hendrickson, W. A. (1996) Nature 384, 484–489[CrossRef][Medline] [Order article via Infotrieve]
  9. Hubbard, S. R. (1997) EMBO J. 16, 5572–5581[CrossRef][Medline] [Order article via Infotrieve]
  10. Nada, S., Okada, M., MacAuley, A., Cooper, J. A., and Nakagawa, H. (1991) Nature 351, 69–72[CrossRef][Medline] [Order article via Infotrieve]
  11. Grgurevich, S., Linnekin, D., Musso, T., Zhang, X., Modi, W., Varesio, L., Ruscetti, F. W., Ortaldo, J. R., and McVicar, D. W. (1997) Growth Factors 14, 103–115[Medline] [Order article via Infotrieve]
  12. Okada, M., Nada, S., Yamanashi, Y., Yamamoto, T., and Nakagawa, H. (1991) J. Biol. Chem. 266, 24249–24252[Abstract/Free Full Text]
  13. Lamers, M. B., Antson, A. A., Hubbard, R. E., Scott, R. K., and Williams, D. H. (1999) J. Mol. Biol. 285, 713–725[CrossRef][Medline] [Order article via Infotrieve]
  14. Ogawa, A., Takayama, Y., Sakai, H., Chong, K. T., Takeuchi, S., Nakagawa, A., Nada, S., Okada, M., and Tsukihara, T. (2002) J. Biol. Chem. 277, 14351–14354[Abstract/Free Full Text]
  15. Sun, G., Ke, S., and Budde, R. J. (1997) Arch. Biochem. Biophys. 343, 194–200[CrossRef][Medline] [Order article via Infotrieve]
  16. Joukov, V., Vihinen, M., Vainikka, S., Sowadski, J. M., Alitalo, K., and Bergman, M. (1997) Biochem. J. 322, 927–935[Medline] [Order article via Infotrieve]
  17. Sun, G., and Budde, R. J. (1997) Biochemistry 36, 2139–2146[CrossRef][Medline] [Order article via Infotrieve]
  18. Lee, S., Lin, X., McMurray, J., and Sun, G. (2002) Biochemistry 41, 12107–12114[CrossRef][Medline] [Order article via Infotrieve]
  19. Wang, D., Huang, X. Y., and Cole, P. A. (2001) Biochemistry 40, 2004–2010[CrossRef][Medline] [Order article via Infotrieve]
  20. Sondhi, D., Xu, W., Songyang, Z., Eck, M. J., and Cole, P. A. (1998) Biochemistry 37, 165–172[CrossRef][Medline] [Order article via Infotrieve]
  21. Leon, B. C., Tsigelny, I., and Adams, J. A. (2001) Biochemistry 40, 10078–10086[CrossRef][Medline] [Order article via Infotrieve]
  22. Senften, M., Schenker, G., Sowadski, J. M., and Ballmer-Hofer, K. (1995) Oncogene 10, 199–203[Medline] [Order article via Infotrieve]
  23. Sotirellis, N., Johnson, T. M., Hibbs, M. L., Stanley, I. J., Stanley, E., Dunn, A. R., and Cheng, H. C. (1995) J. Biol. Chem. 270, 29773–29780[Abstract/Free Full Text]
  24. Sun, G., Sharma, A. K., and Budde, R. J. (1998) Oncogene 17, 1587–1595[CrossRef][Medline] [Order article via Infotrieve]
  25. Sun, G., Ramdas, L., Wang, W., Vinci, J., McMurray, J., and Budde, R. J. (2002) Arch. Biochem. Biophys. 397, 11–17[CrossRef][Medline] [Order article via Infotrieve]
  26. Cooper, J. A., and MacAuley, A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4232–4236[Abstract/Free Full Text]
  27. Sun, G., and Budde, R. J. (1999) Arch. Biochem. Biophys. 367, 167–172[CrossRef][Medline] [Order article via Infotrieve]
  28. Sondhi, D., and Cole, P. A. (1999) Biochemistry 38, 11147–11155[CrossRef][Medline] [Order article via Infotrieve]
  29. Shekhtman, A., Ghose, R., Wang, D., Cole, P. A., and Cowburn, D. (2001) J. Mol. Biol. 314, 129–138[CrossRef][Medline] [Order article via Infotrieve]
  30. Till, J. H., Ablooglu, A. J., Frankel, M., Bishop, S. M., Kohanski, R. A., and Hubbard, S. R. (2001) J. Biol. Chem. 276, 10049–10055[Abstract/Free Full Text]
  31. Frankel, M., Ablooglu, A. J., Leone, J. W., Rusinova, E., Ross, J. B., Heinrikson, R. L., and Kohanski, R. A. (2001) Mol. Cell. Biol. 21, 4197–4207[Abstract/Free Full Text]
  32. Ablooglu, A. J., Frankel, M., Rusinova, E., Ross, J. B., and Kohanski, R. A. (2001) J. Biol. Chem. 276, 46933–46940[Abstract/Free Full Text]
  33. Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42–52[Abstract/Free Full Text]
  34. Gibbs, C. S., and Zoller, M. J. (1991) J. Biol. Chem. 266, 8923–8931[Abstract/Free Full Text]
  35. Skamnaki, V. T., and Oikonomakos, N. G. (2000) J. Protein Chem. 19, 499–505[CrossRef][Medline] [Order article via Infotrieve]
  36. Skamnaki, V. T., Owen, D. J., Noble, M. E., Lowe, E. D., Lowe, G., Oikonomakos, N. G., and Johnson, L. N. (1999) Biochemistry 38, 14718–14730[CrossRef][Medline] [Order article via Infotrieve]
  37. MacAuley, A., Okada, M., Nada, S., Nakagawa, H., and Cooper, J. A. (1993) Oncogene 8, 117–124[Medline] [Order article via Infotrieve]
  38. Okada, M., and Nakagawa, H. (1988) Biochem. Biophys. Res. Commun. 154, 796–802[CrossRef][Medline] [Order article via Infotrieve]
  39. Szustakowski, J. D., and Weng, Z. (2000) Proteins 38, 428–440[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. A. Lieser, J. Shaffer, and J. A. Adams
Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk
J. Biol. Chem., December 8, 2006; 281(49): 38004 - 38012.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. A. Lieser, C. Shindler, B. E. Aubol, S. Lee, G. Sun, and J. A. Adams
Phosphoryl Transfer Step in the C-terminal Src Kinase Controls Src Recognition
J. Biol. Chem., March 4, 2005; 280(9): 7769 - 7776.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Lee, X. Lin, N. H. Nam, K. Parang, and G. Sun
Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase
PNAS, December 9, 2003; 100(25): 14707 - 14712.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/26/24072    most recent
M210596200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, X.
Right arrow Articles by Sun, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, X.
Right arrow Articles by Sun, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.