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Posttranslational Protein Modifications

2006; Elsevier BV; Volume: 5; Issue: 10 Linguagem: Inglês

10.1074/mcp.r600009-mcp200

1535-9484

Karl E. Krueger, Sudhir Srivastava,

Chronic Lymphocytic Leukemia Research

Over the last several years major advances in sensitive high throughput technologies have been made in the fields of genomics and proteomics. The hunt for diagnostic and prognostic cancer biomarkers exploits these recent technology platforms. Although the recent developments and use of genomics and proteomics offer much promise in the search for molecular markers of early stage cancers, these methods are inadequate to probe the dynamic nature of signaling processes that cells exhibit during their transformation to become neoplastic. The diverse realm of posttranslational modification (PTM) 1The abbreviations used are: PTM, posttranslational modification; CFG, Consortium for Functional Glycomics; EGFR, epidermal growth factor receptor; GP73, Golgi protein 73; HDAC, histone deacetylase; ILK, integrin-linked kinase; PDGFR, platelet-derived growth factor receptor; PI, phosphatidylinositol; PIP3, phosphatidylinositol 3,4,5-trisphosphate; mTOR, mammalian target of rapamycin; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MEKK, MEK kinase; MKK, mitogen-activated protein kinase kinase; JNK, c-Jun NH2-terminal kinase; JAK, Janus kinase; STAT, signal transducers and activators of transcription; CDK, cyclin-dependent kinase; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; PTEN, phosphatase and tensin homolog. of proteins encompasses many of the critical signaling events occurring during neoplastic transformation. PTMs offer a plethora of candidates for biomarker detection that complement discoveries using strictly proteomics or genomics platforms. Furthermore the potential to pharmacologically impede tumor growth by administration of an agent that interrupts a specific PTM driving oncogenic progression has been the basis of numerous clinical trials currently underway. To draw greater attention to the opportunities afforded by innovative research in PTMs, a 2-day workshop was conducted August 2002 in Bethesda, MD. The goals of this meeting were to address several topics where PTMs play roles in cancer progression, consider what technologies can be applied to clinical prevention or detection of cancer, and assess what PTMs could be pursued for development of promising surrogate markers. Since that time some advancement has been made in technological developments to study PTMs, identifying the central roles they play in cancer progression, and determining their amenability as either a cancer biomarker or therapeutic target. A limiting factor in PTM research is that technologies to screen vast numbers of molecules for a particular type of modification are often not available, although recent developments of specific probes and multiplexed platforms may now make broader scale PTM surveying feasible (1Blixt O. Head S. Mondala T. Scanlan C. Huflejt M.E. Alvarez R. Bryan M.C. Fazio F. Calarese D. Stevens J. Razi N. Stevens D.J. Skehel J.J. van Die I. Burton D.R. Wilson I.A. Cummings R. Bovin N. Wong C.H. Paulson J.C. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17033-17038Crossref PubMed Scopus (968) Google Scholar, 2Ge Y. Rajkumar L. Guzman R.C. Nandi S. Patton W.F. Agnew B.J. Multiplexed fluorescence detection of phosphorylation, glycosylation, and total protein in the proteomic analysis of breast cancer refractoriness.Proteomics. 2004; 4: 3464-3467Crossref PubMed Scopus (39) Google Scholar, 3Ivanov S.S. Chung A.S. Yuan Z.L. Guan Y.J. Sachs K.V. Reichner J.S. Chin Y.E. Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells.Mol. Cell. Proteomics. 2004; 3: 788-795Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 4Kenten J.H. Davydov I.V. Safiran Y.J. Stewart D.H. Oberoi P. Biebuyck H.A. Assays for high-throughput screening of E2 and E3 ubiquitin ligases.Methods Enzymol. 2005; 399: 682-701Crossref PubMed Scopus (14) Google Scholar, 5Sheehan K. Calvert V. Kay E. Lu Y. Fishman D. Espina V. Aquino J. Speer R. Araujo R. Mills G. Liotta L. Petricoin E. Wulfkuhle J. Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma.Mol. Cell. Proteomics. 2005; 4: 346-355Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 6Ptacek J. Devgan G. Michaud G. Zhu H. Zhu X. Fasolo J. Guo H. Jona G. Breitkreutz A. Sopko R. McCartney R.R. Schmidt M.C. Rachidi N. Lee S.J. Mah A.S. Meng L. Stark M.J. Stern D.F. De Virgilio C. Tyers M. Andrews B. Gerstein M. Schweitzer B. Predki P.F. Snyder M. Global analysis of protein phosphorylation in yeast.Nature. 2005; 438: 679-684Crossref PubMed Scopus (820) Google Scholar). For the most part, research in PTM as it relates to biomarker discovery has required the study of discreet modifications on specific proteins of importance to cancer biology. This one by one approach clearly takes time, but the rewards are not to be underestimated in terms of application to cancer detection and treatment. This review highlights the areas where PTMs are of prime importance for cancer diagnosis and treatment. Table I lists many of the more prominent examples where PTM of specific proteins has relevance toward these aspects of clinical practice in oncology. This review highlights the major aspects of PTMs currently investigated by many laboratories. By providing this overview, it should be apparent that PTMs are key to understanding cancer biology and thus should receive special attention for applications in translational research. PTMs that are currently most germane to clinical applications in cancer medicine are briefly introduced below.Table IExamples of PTMs on proteins contributing to oncogenesis or being used in the context of a cancer biomarkerPosttranslational modificationProtein localizationNuclearCytosolic, intracellular organellesPlasma membrane, secretedPhosphorylationpRBs, p53, histones, HDACs, STAT-3PTEN, Akt, MAP kinases, death-associated protein kinase, cyclin-dependent kinasesEGFRs, PDGFR, Abl, ILK, osteopontinGlycosylationGP73CD44; galectins; CA125, CA19-9; MUC1, MUC4, MUC16; prostate-specific antigen; osteopontinUbiquitination, sumoylationp53, NF-κB, histones, HDACsInhibitor of apoptosis proteinsPrenylationRas, Rho, BrafG-protein-coupled receptorsMethylationHistones, DNA polymerase βAcetylationp53, GATA transcription factors, histones, HDACs, NF-κB Open table in a new tab The role of phosphorylation in regulating enzyme activity has long been recognized. Little introduction is needed for this form of PTM as its involvement in intermediary cell metabolism is common knowledge. Defined signaling pathways where aberrant regulation of phosphorylation contributes to oncogenesis include receptor tyrosine kinases/PI 3-kinase/Akt/mTOR, receptor tyrosine kinases/Ras/Raf/MEK/ERK, MEKK/MKK/JNK, and JAK/STAT. All of these signaling cascades, where phosphorylation occurs at nearly each step, have profound control in cell growth, survival, apoptosis, or responses to various extracellular signals. The exploitation of these phosphorylation events for diagnostic and therapeutic intervention in cancer treatment has been a rapidly developing area over the last few years. The role of acetylation appears analogous to that of phosphorylation. By virtue of neutralizing surface charges on lysine residues, acetylation can regulate protein function or its association with other proteins. In the special case of histones described in depth later, acetylation affects the nature of the association of this abundant class of proteins with DNA. The importance of methylation in protein function and tumorigenesis is still a field in its infancy. Methylated lysine residues still carry a positive charge and thus apparently have little effect on protein conformation. However, lysines that are di- or trimethylated are not accessible for other forms of modification such as acetylation or ubiquitination. It is this distinguishing property that implicates methylation as being important in the regulation of proteins by others forms of PTM. Activation of GTPases such as Ras, Rho, and G-proteins coupled to cell surface receptors is a feature common to many cancers. An obligatory step in their activation is prenylation of a cysteine residue near the carboxyl terminus conferring membrane association of these proteins. This modification entails covalent thiolation with either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid group serving as the anchor for membrane attachment. Further processing occurs where the terminal three amino acids are removed by a protease, and the resulting carboxyl-terminal, alkylated cysteine residue is subsequently methylated yielding an isoprenylcysteinyl carboxymethyl ester (7Winter-Vann A.M. Casey P.J. Post-prenylation-processing enzymes as new targets in oncogenesis.Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (278) Google Scholar). Following this series of modification reactions, the GTPase protein is anchored to the inner leaflet of the plasma membrane providing the topology necessary for its signal transduction activity. Ubiquitination is a PTM that likely affects all proteins at some point in their life cycle. The most common role ubiquitination plays is in tagging of proteins for degradation via the 26 S proteasome. This signal for degradation usually involves polyubiquitination of a protein. In contrast, monoubiquitination is believed to serve as a regulatory modification of the protein in much the same way phosphorylation regulates protein activity (8Weissman A.M. Themes and variations on ubiquitylation.Nat. Rev. Mol. Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1257) Google Scholar). SUMO1 is a ubiquitin-like protein that likewise can be covalently attached to target proteins presumably serving a modulatory function (9Kim K.I. Baek S.H. Chung C.H. Versatile protein tag, SUMO: its enzymology and biological function.J. Cell. Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google Scholar). The roles ubiquitination and sumoylation play in tumorigenesis are still poorly understood and perhaps underappreciated; however, cases of ubiquitin ligases showing relationships with oncogenesis are now being uncovered (10Beckmann J.S. Maurer F. Delorenzi M. Falquet L. On ubiquitin ligases and cancer.Hum. Mutat. 2005; 25: 507-512Crossref PubMed Scopus (7) Google Scholar, 11Lim M.S. Elenitoba-Johnson K.S. Ubiquitin ligases in malignant lymphoma.Leuk. Lymphoma. 2004; 45: 1329-1339Crossref PubMed Scopus (14) Google Scholar). Nearly all cell surface and secreted proteins are glycosylated. Proper conformational folding of the translated polypeptide chain is facilitated by glycosylation events, and thus protein function is often dependent on or refined by the carbohydrate moieties attached to the polypeptide. Heterogeneity often exists in the multiple oligosaccharide chains attached to a single protein. The structures of these oligosaccharides are dictated by the panel of highly specific glycosyltransferases and glycosidases present in the endoplasmic reticulum and Golgi apparatus of the cell. Alterations in the expression of these enzymes will result in changes of the glycomic profile found on its glycoproteins. Because neoplastic cells show altered transcriptomic profiles, often resembling more embryonic cellular states, the glycome synthetic machinery of the cell is a module often changed by oncogenic transformation. It is perhaps no wonder that many tumor-specific antigens have been discovered to be cell surface carbohydrate structures (12Hakomori S. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines.Adv. Exp. Med. Biol. 2001; 491: 369-402Crossref PubMed Scopus (383) Google Scholar). There are many other forms of PTM that have been identified; however, their application for cancer medicine is not yet readily apparent. It would be worthwhile to list these PTMs as in the near future they may prove valuable in our understanding of cancer biology and how this disease might be combated. These other modifications include disulfide bond formation, myristoylation, proline isomerization, ADP-ribosylation, transglutamination, citrullination, sulfation, and glycosylphosphatidylinositol anchoring. Moreover a distinct but common type of glycosylation on cytoplasmic and nuclear proteins has been identified where N-acetylglucosamine is linked to serine or threonine residues (13Hanover J.A. Glycan-dependent signaling: O-linked N-acetylglucosamine.FASEB J. 2001; 15: 1865-1876Crossref PubMed Scopus (252) Google Scholar). Alterations in gene expression, activation of certain cellular signaling pathways, enhanced proliferation, and dysregulation of cell division or death have long been recognized as hallmarks of cancer progression. PTMs play pivotal roles in all of these activities because it is the chemical modifications of key regulatory or structural proteins that dictate the activation state for most cell physiological events. This section describes a number of well established examples where carcinogenesis is dependent upon select proteins subjected to, or participating in, PTM resulting in the aberrations of cell physiology, structural integrity of cellular components, and control of gene expression. Because the nucleus is the site where genetic information is unfolded to enact transcriptomic programs, PTM of nuclear proteins poses a tangible link with tumorigenesis. Two prominent examples that portray how various PTM mechanisms play central roles in tumor biology are the p16/pRB/cyclin D1 and p19/p53/MDM2 cell cycle control pathways. At least one of these pathways appears to be inactivated in all tumors, and the ability to probe into these specific PTMs is shedding light on early stages of carcinogenesis. The retinoblastoma gene RB-1 is among the first tumor suppressors to be discovered. The protein product of this gene (pRB1) prevents progression of a cell into S phase by binding to transcription factors of the E2F family and repressing genes involved in nucleotide and DNA synthesis (14Sellers W.R. Kaelin Jr., W.G. Role of the retinoblastoma protein in the pathogenesis of human cancer.J. Clin. Oncol. 1997; 15: 3301-3312Crossref PubMed Scopus (236) Google Scholar, 15Donnellan R. Chetty R. Cyclin D1 and human neoplasia.Mol. Pathol. 1998; 51: 1-7Crossref PubMed Scopus (306) Google Scholar). This repressor activity is relieved when cyclin D1 binds cyclin-dependent kinase CDK4 to then phosphorylate pRB1 causing it to dissociate from E2F permitting this transcription factor to be an activator for genes of DNA synthesis. The cyclin-dependent kinase inhibitor p16 is another tumor suppressor of this control pathway that acts to inhibit the activity of the cyclin D1-CDK4 heterodimer. Phosphorylation of pRB1 is thus the key regulatory step in this pathway controlling cell division. Amplification of the cyclin D1 gene or mutations in p16 are often found in tumors contributing to a hyperphosphorylated state of pRB1 and a consequential commitment to continued cell growth (16Baker G.L. Landis M.W. Hinds P.W. Multiple functions of D-type cyclins can antagonize pRb-mediated suppression of proliferation.Cell Cycle. 2005; 4: 330-338Crossref PubMed Scopus (75) Google Scholar, 17Chatterjee S.J. George B. Goebell P.J. Alavi-Tafreshi M. Shi S.R. Fung Y.K. Jones P.A. Cordon-Cardo C. Datar R.H. Cote R.J. Hyperphosphorylation of pRb: a mechanism for RB tumour suppressor pathway inactivation in bladder cancer.J. Pathol. 2004; 203: 762-770Crossref PubMed Scopus (75) Google Scholar). At least two other proteins have been identified in the pRB family, all showing similar mechanisms of modulation by phosphorylation and cyclins in regulating E2F factors (18Gallo G. Giordano A. Are RB proteins a potential substrate of Pin1 in the regulation of the cell cycle?.J. Cell. Physiol. 2005; 205: 176-181Crossref PubMed Scopus (13) Google Scholar). Reduction of pRB2 expression by promoter methylation has been reported recently to contribute to retinoblastoma tumors and non-small cell lung cancer (19Cinti C. Macaluso M. Giordano A. Tumor-specific exon 1 mutations could be the ‘hit event’ predisposing Rb2/p130 gene to epigenetic silencing in lung cancer.Oncogene. 2005; 24: 5821-5826Crossref PubMed Scopus (16) Google Scholar). These findings draw clear parallels with the more widely known roles of aberrant pRB1 activity in many cancers. In a manner similar to that seen with pRB1, p53, another tumor suppressor, is subject to multiple modes of PTM. Normally p53 is maintained at low levels due to ubiquitination by the ubiquitin E3 ligase MDM2 and ensuing degradation; however, upon exposure to certain stressful stimuli, p53 ubiquitination is suppressed leading to formation of an active tetrameric p53 complex triggering regulation of a host of genes controlling DNA repair, apoptosis, and cell cycle arrest (20Bode A.M. Dong Z. Post-translational modification of p53 in tumorigenesis.Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1022) Google Scholar). In tumor cells MDM2 is often amplified thus constitutively supporting p53 degradation preventing normal cellular responses to stress, whereas inhibition of MDM2-p53 association reverses this effect (21Tovar C. Rosinski J. Filipovic Z. Higgins B. Kolinsky K. Hilton H. Zhao X. Vu B.T. Qing W. Packman K. Myklebost O. Heimbrook D.C. Vassilev L.T. Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: implications for therapy.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1888-1893Crossref PubMed Scopus (583) Google Scholar). Recently specific mutations of p53 have been found to foster its hyperubiquitination (22Shimizu H. Saliba D. Wallace M. Finlan L. Langridge-Smith P.R. Hupp T.R. Destabilizing mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo.Biochem. J. 2006; 397: 355-367Crossref PubMed Scopus (28) Google Scholar) revealing a likely mechanism by which p53 mutations contribute to neoplasia. Other forms of PTM that regulate p53 levels and activity are acetylation and phosphorylation. Acetylation appears to prevent ubiquitination, whereas p53 deacetylation promotes its ubiquitination and subsequent degradation. The existence of at least 17 phosphorylation sites contributes to how p53 controls transcriptional activity. The importance of PTMs in gene regulation and carcinogenesis can easily be extended to the vastly diverse set of other nuclear proteins. For example, histone acetyltransferases can act upon numerous transcription factors such as p53, members of the GATA family, many nuclear receptor superfamily members, and a host of co-activators (23Fu M. Wang C. Zhang X. Pestell R.G. Acetylation of nuclear receptors in cellular growth and apoptosis.Biochem. Pharmacol. 2004; 68: 1199-1208Crossref PubMed Scopus (154) Google Scholar). It should not be surprising that many of the enzymes that catalyze PTMs on other proteins are themselves subject to different forms of PTM. For example, histone deacetylases are subject to regulation by phosphorylation and sumoylation (24Sengupta N. Seto E. Regulation of histone deacetylase activities.J. Cell. Biochem. 2004; 93: 57-67Crossref PubMed Scopus (298) Google Scholar). In essence, nearly every nuclear protein is fair game to be modified in some way as a means of modulating its activity. Inherent in this is the diversity of PTMs that remain to be characterized on such a wide ranging population of host proteins and understanding the consequences they dictate for nuclear function and how this leads to oncogenesis. Chromatin structure is a major determinant affecting gene transcription. This is evidenced in a broad sense by the observation that nuclei of neoplastic cells are generally much more euchromatic in nature. The fundamental unit of chromatin is the nucleosome where DNA is wound around a core particle formed by histones. The next level of chromatin organization is arrangement of nucleosomes into a 30-nm chromatin fiber. Transcriptional initiation usually does not occur when promoters are obscured within nucleosomes packed within 30-nm fibers. Chromatin organization is dynamically remodeled, and a major element of this change involves PTM of histones at their carboxyl-terminal tails by acetylation, methylation, and phosphorylation where elaborate mechanisms are utilized by cells to control the states of histone modification. PTM of histones influences the integrity of the chromatin fiber and thus likely serves as a crucial determinant in exposing promoter DNA elements that can then be recognized by transcriptional factors for assembly of a transcriptional complex. The term epigenetics refers in part to the global state of histone modifications as these particular PTMs play a paramount role in gene expression and do not involve alterations in the sequence of the genes themselves. For this reason there is considerable interest in the roles PTMs of histones play in neoplasia. The state of histone acetylation has broad influence on chromatin structure, nucleosome packing, and hence the transcriptional states of specific genes. Two groups of enzymes responsible for regulating the reversible and dynamic state of histone acetylation are histone acetyltransferases and histone deacetylases (HDACs). Because histones contain a high degree of basic amino acids, acetylation serves to neutralize the abundance of positive charges and decrease histone affinity for the phosphate backbone of DNA. Acetylation of histones, in general, leads to transcriptional activation as nucleosomes unpack from the 30-nm chromatin fibers (Fig. 1), and other transcriptional regulatory proteins now gain access to promoter elements on DNA. As most tumors are characterized by nuclei with a higher degree of euchromatin, normally indicative of unpacked chromatin structure, a significant factor contributing to this phenomenon is histone acetylation. The development of a battery of HDAC inhibitors, discussed in greater detail later in this review, highlight the importance of histone acetylation in cancer progression. Methylation is another PTM commonly found on histones having significant roles in chromatin remodeling. Methylation may be a mechanism to prevent acetylation on lysine residues. Histone methyltransferases utilize S-adenosylmethionine as a methyl donor; however, its unmethylated analogue S-adenosylhomocysteine can decrease histone methyltransferase activity. The role diet can play in this process is of interest as the composition of dietary intake, namely folic acid content, can influence the ratio of S-adenosylmethionine:S-adenosylhomocysteine. The link between diet and cancer is widely recognized, and the role of folic acid as a methylation cofactor certainly has broad implications not only for the activity of histone methyltransferases but also other biochemical processes that are dependent on methyl group donors such as DNA mutations arising from misincorporation of dUTP into DNA and methylation of DNA resulting in gene silencing (25Duthie S.J. Narayanan S. Sharp L. Little J. Basten G. Powers H. Folate, DNA stability and colo-rectal neoplasia.Proc. Nutr. Soc. 2004; 63: 571-578Crossref PubMed Scopus (61) Google Scholar). Several of the major PTMs histones can undergo have been discussed, but the list is longer than discussed here. Phosphorylation, ubiquitination, and sumoylation have also been demonstrated. Recently histone lysine demethylases have been discovered posing yet another level of modulation of chromatin remodeling (26Trojer P. Reinberg D. Histone lysine demethylases and their impact on epigenetics.Cell. 2006; 125: 213-217Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Because there are multiple sites on the histone tails that can accommodate various forms of PTM, some effort has been devoted to understanding how modifications on defined amino acids affect gene expression or chromatin ultrastructure (27Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7666) Google Scholar). These modifications are also likely to attract specific nuclear proteins implementing the assembly of transcriptional complexes with discrete genes. A compelling hypothesis remaining to be tested is whether any particular patterns of histone modifications can distinguish neoplastic cells from normal cells. Until researchers have robust tools to analyze such complex levels of histone PTMs with a myriad of permutations, this question will remain unanswered. Nevertheless because histones play an integral role in altered gene regulation by tumors, the implications of the importance all these histone PTMs likely contribute in oncogenesis are apparent. Altered regulation of signal transduction pathways often plays principal roles in the growth properties of neoplastic cells. Targeted intervention to correct aberrant signaling is a plausible means to reverse the effects of altered signaling mechanisms in cancer cells. Signaling cascades often involve protein phosphorylation of key intermediary regulatory kinases and metabolic enzymes. Markers to identify aberrant signaling are rapidly expanding in light of their clear relationship to growth of neoplastic cells. The paragraphs that follow present some hallmark cases typifying where protein phosphorylation plays some well established roles in neoplastic growth. Deregulation of the pathways using phosphatidylinositol 3,4,5-trisphosphate (PIP3) as a second messenger is common to many types of tumors. The tumor suppressor PTEN is a phosphatase that dephosphorylates PIP3 to phosphatidylinositol 4,5-bisphosphate serving to keep signaling by this second messenger in check. The activity and stability of PTEN is dependent upon phosphorylation near its carboxyl terminus (28Leslie N.R. Downes C.P. PTEN function: how normal cells control it and tumour cells lose it.Biochem. J. 2004; 382: 1-11Crossref PubMed Scopus (347) Google Scholar). In its dephosphorylated state, PTEN is activated to enzymatically degrade PIP3, but PTEN is also quite unstable in the dephosphorylated state, apparently subject to proteasomal degradation. When it is phosphorylated, PTEN recruits binding of another tumor suppressor, PICT-1, conferring greater stability and protection from degradation (29Okahara F. Ikawa H. Kanaho Y. Maehama T. Regulation of PTEN phosphorylation and stability by a tumor suppressor candidate protein.J. Biol. Chem. 2004; 279: 45300-45303Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Approximately 20% of tumor-associated PTEN mutations disrupt the phosphorylation sites at the carboxyl-terminal region resulting in rapid degradation of this critical enzyme. Because many protein kinases participate in signal transduction cascades, great attention has been drawn to tyrosine kinase inhibitors in relation to therapeutic treatment of cancer. The epidermal growth factor receptor (EGFR) subfamily, platelet-derived growth factor receptor (PDGFR), and c-Kit receptor among others all belong to the tyrosine kinase superfamily. Upon dimerization these receptors autophosphorylate to promote binding and tyrosine phosphorylation of other intracellular signaling proteins such as Src, phospholipase Cγ, and PI 3-kinase. Downstream cascade signaling events can then recruit a multitude of pathways that regulate cell growth or apoptosis. These pathways include the participation of such protein kinases as mitogen-activated protein (MAP) kinases and protein kinase B/Akt. Cytokine receptors represent another group of tyrosine kinases known as the Janus kinases or JAKs often implicated in cancer. Following autophosphorylation these kinases bind and phosphorylate a group of latent regulatory proteins termed STATs, which translocate to the nucleus to activate transcription of certain genes that can influence cell proliferation and survival. Integrin-linked kinase (ILK) has signaling properties that tie integrins and growth factors to downstream pathways such as protein kinase B/Akt phosphorylation; activation of β-catenin, cyclin D1, and AP-1 pathways; and expression of matrix metalloproteinase-9, which functions to degrade extracellular matrix to promote the invasion by cancer cells (30Persad S. Dedhar S. The role of integrin-linked kinase (ILK) in cancer progression.Cancer Metastasis Rev. 2003; 22: 375-384Crossref PubMed Scopus (147) Google Scholar, 31Yoganathan N. Yee A. Zhang Z. Leung D. Yan J. Fazli L. Kojic D.L. Costello P.C. Jabali M. Dedhar S. Sanghera J. Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties.Pharmacol. Ther. 2002; 93: 233-242Crossref PubMed Scopus (53) Google Scholar). ILK expression is elevated in several cancers so targeted inhibition of this enzyme may provide a mechanism to treat cancer. ILK inhibitors have been reported to increase survival in a rat orthotopic model of pancreatic cancer (32Yau C.Y. Wheeler J.J. Sutton K.L. Hedley D.W. Inhibition of integrin-linked kinase by a selective small molecule inhibitor, QLT0254, inhibits the PI3K/PKB/mTOR, Stat3, and FKHR pathways and tumor growth, and enhances gemcitabine-induced apoptosis in human orthotopic primary pancreatic cancer xenografts.Cancer Res. 2005; 65: 1497-1504Crossref PubMed Scopus (120) Google Scholar). Because of the host of interactions ILK poses in regulating cell adhesion and extracellular matrix interactions with different signaling pathways, the potential of this protein kinase as a biomarker and therapeutic target should be investigated further. Activation of MAP kinase pathways in tumors stimulates cellular proliferation. Upstream r