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II. SRC TYROSINE KINASE AND OSTEOPOROSIS

II. SRC TYROSINE KINASE AND OSTEOPOROSIS

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Figure 2 Depiction of the active (‘‘open’’) and inactive (‘‘closed’’) conformations of Src kinase based on the analysis of x-ray structures of c-Src tyrosine

kinase crystallized in its inactive state [7]. The stabilization of the inactive

conformation is influenced by multiple events including intramolecular binding

of the tyrosine-phosphorylated C-terminus tail to the SH2 domain as well as

interactions between the SH3 domain and the SH2–kinase linker. CT, C-terminal;

NT, N-terminal.



process that involves both bone degradation (resorption) and bone formation. Aberrantly high levels of bone resorption are associated with this

disease, which effects a net decrease in bone density and volume, resulting

in fragile, brittle bones that are subject to premature breaks and fractures

[8]. The most compelling evidence that Src is intimately involved in bone

remodeling comes from genetically engineered Src knockout mice. In these

Src (–/–) mice, the only major phenotype observed is excessive bone



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



formation; a condition termed osteopetrosis (the opposite of osteoporosis). This suggests that selective inhibition of Src, as a therapeutic treatment

for osteoporosis, may shift the bone microenvironment from a state of

perpetual bone degradation to one of normal bone turnover without

deleteriously affecting other Src-associated cellular processes in the body.

The rationale for Src’s involvement in bone processes becomes

apparent when the Src knockout effects are examined at the cellular level

of an osteoclast. Osteoclasts are multinucleated cells that are responsible

for bone resorption. Two different osteoclasts, a wild-type (normal) and

a Src (–/–) osteoclast, are shown schematically in Figure 3. The wild-type

cell shows the characteristics of a bone-resorbing osteoclast, including a

ruffled border and ‘‘pit’’ formed by the bone-degrading actions of an

active osteoclast. These features are absent in the Src (–/–) knockout

osteoclast, albeit they are still viable and adhere to bone. In 2000

(Marzia et al. and Amling et al.) it was suggested that Src plays a

negative regulatory role in osteoblasts (cells that are responsible for the



Figure 3 The effect of an Src (–/–) knockout in mice as shown by differences in

function and appearance of wild-type and Src-minus osteoclasts. The Src-minus

osteoclasts lack the ruffled borders of a normal, resorbing osteoclast, but are

viable and can adhere to bone. (From Ref. 8.)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



formation of bone) as shown by enhanced bone formation and osteoblast differentiation rates in Src (–/–) mice [8]. Together, these data

provide complementary, mechanistic evidence to validate Src as a viable

therapeutic target for the treatment of bone diseases such as osteoporosis.



III. SH2 DOMAINS AND PHOSPHOPEPTIDE BINDING

The question of whether ligand binding specificity exists among SH2

domains was addressed, quite elegantly, by Cantley et al. [6,9], who used

synthesized combinatorial libraries of pTyr-containing peptides. A majority of the SH2 binding affinity in pTyr-containing peptides can be

attributed to a four-amino acid region represented by the sequence pTyrAaa-Bbb-Ccc. However, binding specificity exists in the three amino acids

directly C-terminal to the pTyr (pY) group, referred to sequentially as

pY+1 (Aaa), pY+2 (Bbb), and pY+3 (Ccc). The preferred pY+1,

pY+2, and pY+3 amino acids for various SH2-containing proteins are

listed in Table 3; the first amino acid listed for each position represents the

most preferred. For Src SH2 this sequence is pTyr-Glu-Glu-Ile. Such

sequence specificity among SH2-containing proteins provides a rationale

for the differentiation of their associated signal transduction pathways

in cells.

The successful design of small molecules to interact with a protein

binding surface is markedly enhanced by an understanding of the target’s

three-dimensional structure, preferably in the context of a bound ligand.

In this regard, early x-ray structures of pTyr-containing peptides bound

to Src SH2 [10,11] paved the way for the discovery of peptide, peptidomimetic, and nonpeptide ligands and the determination of their complexed structures with Src SH2 [12–14] or the highly homologous Lck

SH2 [15,16]. In a landmark paper, Waksman, Kuriyan, and their

colleagues reported [11] the first x-ray structure of a high affinity phosphopeptide (Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu) bound Src

SH2 (KD = 3–6 nM), which uncovered key protein interactions with the

pTyr-Glu-Glu-Ile sequence. In particular, this x-ray structure shows the

bound phosphopeptide oriented perpendicular to a central h sheet and

interacting with two major binding regions of the Src SH2 domain,

namely, one for the ligand pTyr (pY pocket) and another for the ligand

Ile (pY+3 pocket), to provide what has been described as a ‘‘twopronged’’ binding mode. The pY pocket is characterized mainly by



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Table 3 SH2 Specificity for Phosphotyrosine-Containing Peptides

-Asp-Gly-[pTyr-Aaa-Bbb-Ccc]-Ser-Pro(pY)(pY+1)(pY+2)(pY+3)

SH2 Domain



Aaa



Bbb



Ccc



Glu, Asp, Thr

Glu, Thr, Gln



Glu, Asn, Tyr

Glu, Asp



Ile, Met, Leu

Ile, Val, Met



Glu, Thr, Met

Gln, Thr, Glu

Thr

Gln, Tyr, Val



Asn, Glu, Asp

Glu, Gln, Thr

Thr

Asn



Pro, Val, Leu

Thr

Ile, Leu, Met

Tyr, Gln, Phe



Leu, Ile, Val

Val, Ile, Leu

Met, Ile, Val

Ile, Glu, Tyr



Glu, Asp

Ile, Leu







Leu, Ile, Val

Pro, Val, Ile

Met

Ile, Leu, Met



a



Group 1A

Src

Lck

Group 1Ba

Abl

Syk (N-SH2)

Syk (C-SH2)

Grb2

Group 3b

PLCg (N-SH2)

PLCg (C-SH2)

p85 (N-SH2)

SHC

a



Group 1 contains Tyr or Phe at hD5.

Group 3 contains Ile, Cys, or Leu at hD5.

Sources: Refs. 6, 9.

b



electrostatic interactions, while the pY+3 pocket involves interactions

that are mostly hydrophobic.

Figure 4 represents the specific Src SH2 binding interactions with

pTyr-Glu-Glu-Ile sequences, as interpreted from x-ray structures [10,11].

The major intermolecular interactions in the pY pocket involve the

phosphate oxygens of the ligand pTyr side chain with the conserved basic

residues Arg158 and Arg178 of Src SH2. It is noted that Arg178 mutation

results in essentially a total loss of binding affinity [17]. Additional

intermolecular hydrogen-bonding interactions are also observed with

Ser180, Thr182, and the backbone NH of Glu181, whereas a hydrophobic

contact occurs between the alkyl side chain of Lys206 and the phenyl ring

of the ligand pTyr residue. The two adjacent glutamic acid residues

(pY+1 and pY+2) form relatively weak interactions (electrostatic and

hydrophobic) with the protein, albeit their extend side chain conformations (oriented away from each other) serve to align and rigidify the

peptide backbone. This is an important feature from a drug discovery

perspective and can be used in the design of rigid, nonpeptide templates



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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