the mammalian nonreceptor tyrosine kinases ( nrtks ) are divided into ten families : src , abl , jak , ack , csk , fak , fes , frk , tec , and syk     . in addition to their tyrosine kinase catalytic domains , they all contain noncatalytic domains that are important in enzyme regulation and substrate recognition . like all eukaryotic protein kinases , nrtk catalytic domains have an n - terminal lobe ( n - lobe ) that contacts atp , and a larger c - terminal lobe ( c - lobe ) . the activation state of the tyrosine kinases depends on the orientation of an alpha helix ( c ) located in the n - lobe . in the active conformation , the c helix projects inward toward the atp - binding site . the conformation of a flexible segment in the c - lobe ( the activation loop ) also has a key role in the regulation of the enzyme activity . the regulatory importance of the phosphorylation of the activation loop varies in the different families of nrtks . for instance , src activation is strongly dependent on autophosphorylation , while csk lacks the autophosphorylation site . the contributions of the noncatalytic domains to enzyme regulation are well understood in the case of the src - family of nrtks . these enzymes have an n - terminal myristoylation site followed by an sh3 domain , an sh2 domain , and a kinase domain . two tyrosine residues are important for their activity : tyr 416 in the activation loop and tyr 527 in the c - terminal tail . structural studies showed the spatial arrangement of src domains in the inactive state , in which the noncatalytic domains mediate intramolecular interactions that maintain the enzyme in a closed , inactive conformation . in this conformation , the sh3 domain is bound to a proline - rich helix located between the sh2 domain and the kinase domain , and the sh2 domain is bound to the phosphorylated tyr 527     [ 7 , 8 ] . activating signals provided by protein - protein interactions that destabilize these intramolecular interactions may be of three types : the binding of the sh3 domain to a proline - rich motif ( sh3 binding domain )     ; the dephosphorylation of the tyr527 in the c - terminal tail ; the binding of the sh2 domain to other phospho - tyr containing proteins . in addition , the noncatalytic domains provide surfaces for different types of interactions that couple the activation of the catalytic domain to substrate recognition     [ 2 , 12 , 13 ] . for example , abl kinases have similar arrangements of domains as src - family kinases , but the regulatory mechanisms are different ; this has important implications for the design of pharmacological inhibitors . in both src- and abl - family nrtks , the sh3 domain interacts with a poly - proline helix in the linker region between the sh2 domain . in contrast to src , the sh2 domain of abl interacts in a phospho - tyr independent manner with the c - terminal lobe of the kinase domain . an additional difference is that an n - terminal cap and a myristoyl group wrap around the base of the kinase domain of abl and stabilize the inhibited structure     . importantly , the conformation of the inhibited activation loop is unique to the c - abl catalytic domain . the inhibitor imatinib ( gleevec ) has been particularly successful due to its ability to target this conformation     . as new structures of nrtks structural studies have illuminated the contributions of noncatalytic domains to the regulation of syk-    [ 3 , 5 ] , csk-     [ 6 , 1517 ] , and fes - family kinases . for instance , in csk and fes kinases , interactions between the sh2 and the catalytic domains stabilize the active conformation . in general , the noncatalytic domains of all nrtks the domain architecture of human ack1 ( also called tnk2 ) is shown in figure 1 ( top ) . ack1 is a 120  kda protein with an n - terminal sterile alpha motif ( sam ) domain     , a kinase domain , an sh3 domain , and a cdc42/rac - interactive domain ( crib )     . several functionally relevant regions have been identified in the c - terminal region of ack1 : multiple proline - rich sequences , a clathrin - binding motif     , an ubiquitin binding domain     , and a region that shares a high homology with mig6     . they are the only nrtks with the sh3 domain located c - terminal to the kinase domain , and they are also the only tyrosine kinases with a crib domain . in view of its unique domain composition , the regulatory features of ack1 are likely to differ from those observed in other nrtks . ack1 belongs to a family of nrtks that includes human tnk1 , and homologous proteins in mouse , cow , drosophila melanogaster , and caenorhabditis elegans     [ 23 , 24 ] . although all the members share the overall domain architecture shown in figure 1 ( i.e. , kinase domain and sh3 domain ) , there are major differences in the c - terminal regions of the proteins . furthermore , tnk1 , dack ( from drosophila ) , and kos1 ( from mouse ) lack crib domains     . ack1 , the first member of the ack family , was cloned from a human hippocampal expression library by its ability to bind gtp - bound cdc42 specifically and not rac1 or rhoa     . the gene encoding ack1 is located on chromosome 3q29 in humans , a region that is associated with recurrence of prostate cancer and is a predictor of metastatic relapse in breast cancer     . tnk1 ( thirty - eight - negative kinase 1 ) was originally cloned from hematopoietic stem / progenitor cells from umbilical cord blood in humans     . tnk1 is a member of the ack family with a predicted size of 72  kda and the same domain architecture as ack for the region common to both family members     . in contrast with the other members of the ack family ( namely , ack1/tnk2 , ack2 , and dpr2 ) , but similarly to dack , tnk1 , and kos1 lack the crib domain     [ 24 , 27 ] . tnk1 , was found to be specifically expressed in umbilical blood and not other hematopoietic tissues and to interact with phospholipase c-1 through its proline - rich region     . it has been reported that during embryonic development , tnk1 blocks nf-b activation and thereby promotes apoptosis mediated by the tnf signaling pathway . kos1 is a 47  kda protein that was cloned from differentiating murine embryonic stem cells     . the kos1 gene is located in chromosome 11 in mice and it is thought to be a murine splice variant of the human tnk1     . ack2 has a predicted size of 83  kda and contains the same basic domain structure as ack1 . ack2 is thought to be an isoform of ack1 that is generated by alternative splicing and is not encoded by a different gene     . the ack2 c - terminal region contains a 15-residue insert and has 344 fewer amino acids than ack1     . in humans there is no evidence for the existence of an ack2 homologue     [ 18 , 25 ] . however , a number of the experimental studies on ack ( activation pathways , biochemical properties , and biological roles ) focused on ack2 , and they will be discussed throughout this paper .    in drosophila the roles of dpr2 and dack have not been dissected but they are thought to be different because dpr2 has a cdc42-binding domain , while dack lacks one . the tyrosine kinase activity of dack has been implicated downstream of cdc42 in the pathway that leads to dorsal closure of the epidermis during embryogenesis     . in c. elegans , ark-1 ( a ras - regulating kinase 1 ) , a cytoplasmic tyrosine kinase that is related to ack1 , acts as a negative regulator of egfr signaling . in a yeast two - hybrid analysis , ark-1 interacts with the grb2 homologue , sem5     . the degree of conservation of the full - length sequences varies among the family members . the common core that is conserved in all of the ack family members spans from the n - terminus to the sh3 domain . the amino acid identity of this core ( compared with ack1 ) ranges between 100% for ack2 and 29% for kos1 ( figure 1 ) . the ack1 kinase domain itself shares  40% amino acid identity with other nonreceptor and receptor tyrosine kinases . phylogenetic analyses of human tyrosine kinase catalytic domains place ack1 near epidermal growth factor receptor ( egfr ) family kinases     [ 32 , 33 ] . as described below , ack1 and egfr may also share aspects of enzymatic regulation . expression of ack1 has been demonstrated in several tissues in mammals , with the highest expression in spleen , thymus , and brain     . a number of stimuli , including egf , pdgf     , bradykinin     , agonists of the m3 muscarinic receptor     , and integrin - mediated cell adhesion     [ 35 , 36 ] promote phosphorylation and activation of ack1 . upon egf stimulation , ack1 phosphorylates and interacts with the rho - family gtpase cdc42 and the adaptor protein grb2 and these interactions are essential for this pathway     . the association of ack1 with grb2 has been shown in several studies through different approaches . in vitro studies showed that ack1 associates with several sh3 domains , including the sh3 domain from grb2     . ack1 association with multiple receptor tyrosine kinases such as axl , ltk , and alk is also dependent on grb2     . ack is involved in integrin 1-mediated signaling pathways that modulate processes such as cell migration , cell adhesion , and cell spreading . ack2 is present in a complex with integrin 1 ; it is activated by cell adhesion to a fibronectin - coated surface in a cdc42-dependent manner and it activates the phosphorylation of jnk     . in response to cdc42 activation , ack1 mediates the phosphorylation of p130cas to promote cell migration     [ 35 , 40 ] . ack1 is found in a complex with cas , crk , and cdc42 in collagen - stimulated cells . a signaling complex composed of cdc42 , ack1 , and cas also participates in the regulation of melanoma cell spreading by melanoma chondroitin sulphate proteoglycan ( mcsp ) , a proteoglycan that is expressed on the surface of melanoma cells     . the expression of ack2 in hela cells increases cell migration in a mechanism that also involves p130cas and crkii . thus , one of the physiological functions of ack1 appears to be related to the regulation of cell adhesion and cell migration . these studies also suggest that ack1 activity is involved in the development of metastatic potential of transformed cells . several studies have focused on the role of ack1 in egf receptor trafficking and dynamics . egfr stability and recycling are regulated by interactions between ack1 and multiple protein partners including ubiquitin     , clathrin heavy chain     [ 20 , 42 ] , and sh3px1     [ 43 , 44 ] . in hela cells , overexpression of ack1 inhibits egfr endocytosis and causes the accumulation of egf in internal structures of endocytic origin , suggesting that ack1 is involved in the intracellular sorting of activated egfr . the knock - down of ack1 increases the rate of recycling i - egf to the plasma membrane and reduces the degradation of i - egf , suggesting that ack1 promotes degradation of egfr     . in cos7 cells , overexpression of ack1 promotes egfr degradation by binding to the ubiquitinated egf receptor     . increased stability of ack1 caused by a somatic mutation ( s985n ) in the ubiquitin binding domain ( uba ) results in defective egfr downregulation and sustained activation of downstream signaling . in addition to the effect of ack1 on receptor stability and dynamics , there is a reciprocal effect on ack1 stability . after receptor stimulation by egf , ack1 is recruited to an egfr complex     [ 21 , 47 ] . this complex promotes the activation of ack1      and egfr degradation     [ 21 , 45 , 46 , 48 ] as well as ack1 turnover     [ 49 , 50 ] . the egf - induced degradation of ack1 is signaled by ubiquitination     [ 49 , 50 ] . , the e3 ubiquitin ligase nedd4 - 2 was shown to ubiquitinate the uba domain of ack1 and induce its degradation by proteasomes     . in a second report , the e3 ligase nedd4 - 1 was demonstrated to ubiquitinate the ack1 sam domain , while the deletion of the uba domain enhanced ubiquitination . in these experiments , the degradation of ack1 was mediated by lysosomes rather than by proteasomes     . in both cases , the ww domains of the e3 ligases bound to a conserved ppxy motif in ack1 . the crystal structure of the isolated kinase domain of ack1 has been solved in both phosphorylated and unphosphorylated forms . in these structures , the activation loop conformations are similar and do not occlude the substrate - binding site , suggesting that the phosphorylation of the activation loop may not play a dramatic stimulatory role . the only other structural information available is for the isolated crib domain of ack1 in a complex with cdc42     . in the absence of a structure of full - length ack1 , biochemical experiments have helped to reveal how each of the domains contributes to ack1 function . a major autophosphorylation site has been mapped to the y284 in the activation loop     . a construct containing residues 1- 476 , corresponding to the n - terminus , the kinase domain , the sh3 domain , and the crib domain ( nt - kinase - sh3-crib ) is the longest construct that has been purified to homogeneity     . this construct is activated , although modestly ( 3-fold ) , by the phosphorylation of y284 . transformed cell lines , y826 , y856 , and y857 of ack1 are phosphorylated      ( figure 2 ) . the phosphorylation of y826 was also identified in a study of phosphorylated proteins downstream of insulin signaling . y857 of ack1 was identified in a mass spectrometry study in which protein phosphorylation events downstream of the egfr - family member her2 were analyzed     . in addition , the phosphorylation of y518 was identified in a study of global tyrosine phosphorylation patterns in cancer cells     . interestingly , y826 , y857 , and y858 are located in the mig6-homology region ( mhr ) , a portion of ack1 that participates in inhibitory intramolecular interactions     . it is possible that the mhr of ack1 is a substrate for oncogenic bcr - abl , or for insulin and egf receptors , resulting the release of autoinhibition . relatively little information is available about the regulatory mechanisms of ack1 and the roles that each domain plays in ack1 regulation . the purified nt - kinase - sh3-crib polypeptide is active in vitro and can be used in enzymatic assays     . the peptide substrates that are the most efficiently phosphorylated by this construct are eaiyaapfakkkg ( abl consensus substrate ) or kviydfiekkkkg ( peptide derived from wasp tyrosine phosphorylation site ) . in addition to this tyrosine kinase activity , ack1 acts as a dual - specificity kinase towards wasp and the phosphorylation of a serine residue of wasp ( wiskott - aldrich syndrome protein ) increases the ability of wasp to polymerize actin . ligands for the sh3 domain and the crib domain bind to this construct , but fail to activate it , although addition of cdc42 in vivo does increase ack1 y284 phosphorylation     . residues important for the interaction between ack1 and cdc42 have been identified from the nmr structure of the complex formed by a peptide spanning the residues 548489 of ack1 ( crib domain is 548475 ) and cdc42     . one way of studying the role of each domain in ack1 regulation is to assess the effects of point mutations on the enzyme 's phosphorylation state ( figure 2 ) . a mutation that occurs in ovarian endometrioid carcinoma ( e346k ) has been helpful to gain insight into the role of the mig6 homology region ( mhr ) . these studies have suggested that there is an autoinhibitory interaction between the c - lobe of ack1 kinase domain and the mhr     . mutations such as v365r ( in the kinase domain ) or f820a and e346k ( in the mhr ) destabilize this conformation and activate ack1     . ack is activated by additional cancer - associated mutations located in the sam domain ( r34l and r99q ) , and in the sh3 domain ( m409i ) , but the mechanism is not clear . several studies have produced a constitutively active ack1 by the point mutation of l487f in the crib domain     [ 56 , 57 ] . the position of this mutation is thought to be analogous to l107f in pak1 , which is known to prevent an autoinhibitory interaction . however , it is not known whether the crib domain of ack1 participates in autoinhibition . a mutation designed to prevent the binding of the sh3 domain to its ligand ( w426k ) was reported to produce activation of ack1     . in contrast , a point mutation that disrupts cdc42 binding ( h464d ) reduced ack1 autophosphorylation     . the deletion of the n - terminal sam domain reduced ack1 autophosphorylation and kinase activity     [ 18 , 64 ] . the sam domain has the potential to drive ack1 to the plasma membrane     [ 18 , 64 ] and to form dimers or multimers that increase the local concentration in order to stimulate ack1 activation . interestingly , the deletion of the ack1 sam domain also prevented egf - induced ack1 ubiquitination , consistent with the requirement of the sam domain for activation   and with the observation that ack1 is ubiquitinated after activation     . these results suggest that the individual domains of ack1 are involved in regulatory interactions that could be either inter- or intramolecular . the downregulated state of ack1 involves the intramolecular interaction of the mhr with the kinase domain ( figure 3(a ) ) . as a result of a stimulatory signal , provided by activated egfr , one or more tyrosine residues located in the ack1 mhr are phosphorylated ( figure 3(b ) ) . in addition , the sam domain of ack1 is required for full autophosphorylation and activation of ack1 . it is not clear whether the sam domain participates in autoinhibition but as noted above , it is involved in subcellular targeting and required for maximal enzymatic activity . the domain composition of ack1 is complex and it is likely that in the future , additional interactions ( both intra - and intermolecular ) will be described that will explain in more detail the molecular mechanisms of ack1 regulation and signaling . ack1 has been implicated in several stages and several types of cancer ; this topic has been reviewed recently in . early experiments demonstrated that a peptide derived from the ack1 crib domain blocked the ras - mediated transformation of nih-3t3 cells     . ack1 expression was later shown to be required for the survival of cells transformed by v - ras .   in a study of ~180 advanced - stage cancers , 9% of the lung tumors and 14% of the ovary tumors had an amplification of the ack1 gene on chromosome 3 . in addition , 42% of the aggressive lung tumors and 77% of metastatic hormone refractory prostate tumors showed overexpression of the ack1 mrna . ack1 overexpression enhanced migration of human mammary epithelial cells ( hmecs ) and increased metastasis in the mouse mammary 4t1 breast cancer system     . moreover , activation of ack1 by overexpression of ack1 produced several epithelial - to - mesenchymal transition ( emt ) phenotypes in the human breast cancer cell line mda - mb-231 . in renal cancer cells , activation of ack1 by a somatic point mutation also resulted in emt phenotypes . thus , activation of ack1 is associated with poor prognosis and metastatic phenotypes in human tumors     . in addition , the knockdown of ack1 in breast cancer cells inhibits cell migration     . this suggests a role for ack1 as a metastasis determinant , perhaps as a modifier of other pre - metastatic lesions . the mechanistic basis of ack1 's involvement in prostate tumorigenesis has been explored in detail . in the cultured prostate cancer cell line lncap , the androgen receptor ( ar ) is a transcriptional activator that plays important roles in the development of advanced stage ( androgen - independent ) prostate cancer . activated ack1 has been shown to phosphorylate and activate ar function and to promote the progression of prostate cancer     . in addition , ack1 tyr284 phosphorylation was recently found to correlate with disease progression and proposed to be prognostic of progression of prostate cancer .   a large - scale study that screened cancer tissues for somatic mutations identified four missense mutations in ack1     . in lung adenocarcinoma and ovarian carcinoma , two mutations located in the n - terminus ( r34l and r99q , resp . ) were identified ; in ovarian endometrioid carcinoma , a mutation in the kinase catalytic domain ( e346k ) was identified ; and in lung adenocarcinoma , a mutation in the sh3 domain ( m409i ) was found ( figure 2 ) . the four cancer - associated mutations activate ack1 and promote anchorage independent growth and cell migration     . an additional somatic mutation found in renal cancer , s985n , located in the uba domain of ack1 , increases ack1 stability and enhances oncogenic signaling through egfr . the functions of ack1 in cancer cells appear to be of two types : kinase - independent and kinase - dependent . the effect of ack1 on cell proliferation is independent of ack1 activity     . in a phosphoproteomic study of the action of the antitumor drug dasatinib in lung cancer , this study highlighted a possible scaffolding function of ack1 in the context of control of cell growth     . in contrast , the activation state of ack1 appears to be important in processes that involve enhanced cell motility , that are crucial in the metastatic stage of cancer progression and correlate with poor prognosis     [ 25 , 58 , 67 ] . in breast cancer , ack1 phosphorylates and promotes the activation of akt , an important mediator of signaling pathways that lead to transformation . a number of ack1 substrates have been identified in normal cells as well as cancer cells . these results , together with the enhanced migration of cells overexpressing ack1      or expressing an activated form of ack1     , suggest that ack1 plays a role in the regulation of cell adhesion and migration . ack1 also phosphorylates androgen receptor ( ar ) , increasing the levels of phosphorylated ar in prostate cancer and promoting ar - mediated gene transcription     [ 68 , 69 ] . a comprehensive list of known ack1 substrates and interacting proteins was recently published , and it includes diverse molecules involved in signaling such as receptor tyrosine kinases ( merk , egfr , pdgfr , axl , alk , and ltk ) , membrane proteins ( integrin , mcsp ) , adaptor molecules ( grb2 , hsh2 ) , nucleotide exchange factors ( dbl ) , a transcription activator ( ar ) , a tumor suppressor ( wwox ) , and a proto - oncogene ( akt / pkb ) . other ack1 interactors include proteins involved with vesicle dynamics ( clathrin , snx9 ) and cytoskeleton remodeling ( wasp ) . a number of sh3 containing proteins , such as hck     , grb2 , and snx9     [ 44 , 72 ] have been identified as ligands for the proline rich region of ack1 . recently , the e3 ubiquitin ligases nedd4 - 1 and nedd4 - 2 were shown to bind to a ppxy motif of ack1 and to regulate its proteolytic degradation     [ 49 , 50 ] . nonreceptor tyrosine kinases share several common features . the nrtk catalytic domains never occur in isolation ; they are tethered to noncatalytic modular domains that play important roles in enzymatic function . the biochemical properties of the individual noncatalytic domains are similar to those found in other signaling proteins . however , the regulatory mechanisms are diverse and vary between the different families of nrtks . to date , 15 out of the 32 nrtks in the human genome have been shown to have oncogenic potential     . activation of ack1 ( by overexpression or mutation ) promotes migration and emt and correlates with cancer progression . thus , ack1 has emerged as a candidate for anti cancer drug design ; ack1 inhibitors could potentially be used in combination therapies with inhibitors of other tyrosine kinases , such as egfr . knowledge about nrtk regulatory mechanisms can be exploited for the development of specific inhibitory drugs . for example , imatinib inhibits abl ( but not src ) because it binds to a specific conformation of abl that src is not capable of adopting     . moreover , the inhibited conformation of abl appears to be the result of the interplay of the noncatalytic domains     . ack1 is regulated by a novel mechanism that shares basic principles with other nrtks but also has unique features dictated by its unusual domain structure . investigations into the regulatory mechanisms and conformational states of ack1 will be an important prelude to the development of specific ack1 inhibitors .
ack family non - receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties . 
 human ack1 ( activated cdc42-associated kinase ) is ubiquitously expressed and is activated by signals that include growth factors and integrin - mediated cell adhesion . 
 stimulation leads to ack1 autophosphorylation and to phosphorylation of additional residues in the c - terminus . 
 the n - terminal sam domain is required for full activation . 
 ack1 exerts some of its effects via protein - protein interactions that are independent of its kinase activity . in the basal state , 
 ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the c - terminal region . 
 inappropriate ack1 activation and signaling has been implicated in the development , progression , and metastasis of several forms of cancer . thus , there is increasing interest in ack1 as a drug target , and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design .