Intramolecular Regulatory Interactions in the Src Family Kinase Hck Probed by Mutagenesis of a Conserved Tryptophan Residue

LaFevre-Bernt, Michelle; Sicheri, Frank; Pico, Alexander R.; Porter, Margaret; Kuriyan, John; Miller, W. Todd

doi:10.1074/jbc.273.48.32129

Cited by 95 publications

(122 citation statements)

References 33 publications

Supporting

Mentioning

118

Contrasting

Order By: Relevance

“…In support of this idea, in the structure of the active Lck kinase domain, this residue adopts an alternate conformation and does not contact the C helix (Yamaguchi and Hendrickson, 1996). Also, mutagenesis studies support the notion that Trp 260 helps to communicate the position of the SH2 and SH3 domains to the active site (LaFevre-Bernt et al, 1998). Additional residues in the linker may also participate .…”

Section: Structure Of the Unique Domain Of Lckmentioning

confidence: 55%

Structure and regulation of Src family kinases

2004

View full text Add to dashboard Cite

Src family kinases are prototypical modular signaling proteins. Their conserved domain organization includes a myristoylated N-terminal segment followed by SH3, SH2, and tyrosine kinase domains, and a short C-terminal tail. Structural dissection of Src kinases has elucidated the canonical mechanisms of phosphotyrosine recognition by the SH2 domain and proline-motif recognition by the SH3 domain. Crystallographic analysis of nearly intact Src kinases in the autoinhibited state has shown that these protein interaction motifs turn inward and lock the kinase in an inactive conformation via intramolecular interactions. The autoinhibited Src kinase structures reveal a mode of domain assembly used by other tyrosine kinases outside the Src family, including Abl and likely Tec family kinases. Furthermore, they illustrate the underlying regulatory principles that have proven to be general among diverse modular signaling proteins. Although there is considerable structural information available for the autoinhibited conformation of Src kinases, how they may assemble into active signaling complexes with substrates and regulators remains largely unexplored.

show abstract

Section: Structure Of the Unique Domain Of Lckmentioning

confidence: 55%

Structure and regulation of Src family kinases

2004

View full text Add to dashboard Cite

show abstract

“…The bound proteins were separated by SDS-PAGE and analyzed with anti-ACK and anti-GST antibodies. much lower than the value for Hck (60 min Ϫ1 ) (47). One possible explanation for the low activity is that the appropriate substrates have not yet been identified, Of course, we cannot rule out the possibility that the activity of this truncated ACK1 is artificially low because of problems in protein expression or folding.…”

Section: Discussionmentioning

confidence: 78%

Biochemical Properties of the Cdc42-associated Tyrosine Kinase ACK1

Yokoyama

Miller

2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr 284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or Hck) via its C-terminal proline-rich domain. Co-expression of Hck with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for Hck. Our data suggest that Hck is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.Members of the Rho family of small GTP-binding proteins couple extracellular signals to the regulation of cell morphology, adhesion, differentiation, and proliferation (1-5). A number of proteins have been identified as targets for the Rho family proteins Rac and/or Cdc42, including p21-activated kinases (6 -8), Wiscott-Aldrich syndrome proteins (9 -11), mixed lineage kinases (12), and IQGAP (13-16). The ACK family nonreceptor tyrosine kinases (ACK1 and ACK2) associate specifically with Cdc42 and act as Cdc42 effectors in several signaling pathways (6,17,18). ACK1 has been reported to phosphorylate Dbl, a guanine nucleotide exchange factor toward Rho family proteins, thereby promoting Dbl activity. In this way, ACK1 is thought to act as a mediator of EGF 1 signals to Rho family GTP-binding proteins (19). ACK2 mediates cell adhesion signals initiated by integrin ␤ 1 in a Cdc42-dependent manner (20). ACKs interact directly with the clathrin heavy chain and participate in the regulation of receptor-mediated endocytosis (21, 22). However, the physiological functions as well as specific target molecules of ACKs are still incompletely understood.The domain structure of ACK kinases consists of an Nterminal tyrosine kinase catalytic domain followed by an SH3 domain, a Cdc42/Rac interactive binding (CRIB) domain, and a proline-rich region (see Fig. 1...

show abstract

“…Indeed, ACK1 had barely detectable activity against peptides with methionine or phenylalanine at this position, and the optimal substrate reported for ACK1 contains an alanine at this position (19). Interestingly, the reported turnover rate for this peptide is still quite low (k cat ϭ 0.97 min Ϫ1 ) compared with those for other tyrosine kinases such as Hck, which has a k cat of 60 min Ϫ1 against its preferred peptide substrate (35).…”

Section: Resultsmentioning

confidence: 98%

Crystal Structures of the Phosphorylated and Unphosphorylated Kinase Domains of the Cdc42-associated Tyrosine Kinase ACK1

Lougheed

Chen

Mak

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

ACK1 is a multidomain non-receptor tyrosine kinase that is an effector of the Cdc42 GTPase. Members of the ACK family have a unique domain ordering and are the only tyrosine kinases known to interact with Cdc42. In contrast with many protein kinases, ACK1 has only a modest increase in activity upon phosphorylation. We have solved the crystal structures of the human ACK1 kinase domain in both the unphosphorylated and phosphorylated states. Comparison of these structures reveals that ACK1 adopts an activated conformation independent of phosphorylation. Furthermore, the unphosphorylated activation loop is structured, and its conformation resembles that seen in activated tyrosine kinases. In addition to the apo structure, complexes are also presented with a non-hydrolyzable nucleotide analog (adenosine 5 -(␤,␥-methylenetriphosphate)) and with the natural product debromohymenialdisine, a general inhibitor of many protein kinases. Analysis of these structures reveals a typical kinase fold, a pre-organization into the activated conformation, and an unusual substrate-binding cleft.ACK1 (activated Cdc42-associated kinase-1) is a member of a small family of non-receptor tyrosine kinases that bind Cdc42 in its GTP-bound form. Members of the ACK family have been identified in many species, including human (TNK1) (1), cow (ACK2) (2), Drosophila (DAck, DPR2) (3, 4), and Caenorhabditis elegans (ARK1) (5). ACK1 is a multidomain protein, and all family members contain an SH3 1 domain and a C-terminal proline-rich region. ACK family members are largely expressed in brain and skeletal tissue (2), and multiple lines of evidence suggest that they are involved in the regulation of cell adhesion and growth (6), receptor degradation (7), and axonal guidance (3). ACK1 has been implicated as a mediator of epidermal growth factor signaling to Rho family GTP-binding proteins through phosphorylation and activation of guanosine exchange factors such as Dbl, suggesting a role in regulation of the cytoskeleton (8, 9). ACK1 has also been reported to bind to adaptor proteins, suggesting that it is involved in several different signaling pathways. The SH3 domain of ACK1 binds to the proline-rich region of HSH2, an adaptor protein in hematopoietic cells, leading to its phosphorylation (10), whereas the C-terminal proline-rich domain of ACK1 interacts with the adaptor proteins Nck (11) and Grb2 (12), which are involved in cytoskeletal rearrangement (13) and Ras activation (14), respectively. In addition, both DAck and ACK2 interact with the sorting nexin SH3PX1 through their C-terminal domains, leading to its phosphorylation (3, 7), and this ACK2 interaction with SH3PX1 together with clathrin promotes degradation of EGFR (7).The domain architecture of the ACK family is unique compared with other non-receptor tyrosine kinases, and ACK family members are the only tyrosine kinases known to interact with Cdc42 (15). Members of the ACK family are specific for Cdc42 and do not bind the closely related Rac and Rho GTPases (2, 15). The 114-kDa human ACK1 p...

show abstract

Intramolecular Regulatory Interactions in the Src Family Kinase Hck Probed by Mutagenesis of a Conserved Tryptophan Residue

Abstract: Intramolecular interactions between the

Cited by 95 publications

References 33 publications

Structure and regulation of Src family kinases

Structure and regulation of Src family kinases

Biochemical Properties of the Cdc42-associated Tyrosine Kinase ACK1

Crystal Structures of the Phosphorylated and Unphosphorylated Kinase Domains of the Cdc42-associated Tyrosine Kinase ACK1

Contact Info

Product

Resources

About