A Peptide Model System for Processive Phosphorylation by Src Family Kinases

Mp, Scott; Wt, Miller

doi:10.1021/bi001850u

Cited by 51 publications

(53 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As recently demonstrated, the interaction of the p60 src SH3 domain with the ligand sequence of the substrate is important for activation of the catalytic domain and autophosphorylation (41), and the addition of an SH3 domain ligand to a substrate peptide increases its phosphorylation 10-fold via lowering of the K m value of the substrate and kinase activation (42). Thus, putative SH3-and SH2-binding domains present in MLCK-1 N-terminal portion (Fig.…”

Section: Identification Of P60mentioning

confidence: 73%

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Birukov

Csortos

Marzilli

et al. 2001

Journal of Biological Chemistry

148

140

View full text Add to dashboard Cite

The Ca 2؉ /calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210 -214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch ( 20 by Ca 2ϩ /calmodulin-dependent enzyme MLCK is essential for the initiation of nonmuscle and smooth muscle contraction (1, 3, 9 -11). In specific nonmuscle tissues, such as the vascular endothelium, this kinase is known to be involved in endothelial cell migration, cell retraction (12), endothelial cell barrier regulation (13), transendothelial migration of neutrophils (14, 15), and possibly apoptosis (16). The MLCK isoform abundantly expressed in smooth muscle (SM MLCK) generally exists as a 130-to 150-kDa protein that has been well characterized (for review see Refs. 3 and 11). However, Western blot screening of a variety of embryonic and adult smooth muscle and nonmuscle tissues revealed expression of a high molecular weight MLCK variant with electrophoretic mobility in the range of [208][209][210][211][212][213][214]. Garcia and colleagues (20) subsequently sequenced the high molecular weight MLCK isoform cloned from a human endothelial cell cDNA library, revealing an open reading frame, which encodes a protein of 1914 amino acids. Both the low molecular mass (130 -150 kDa) and high molecular mass (208 -214 kDa) MLCK isoforms share essentially identical actin binding, MLC binding, catalytic, and Ca 2ϩ /CaM-regulatory domains. The extreme C-terminal kinase-related protein (KRP) domain, which binds myosin, is contained within both EC MLCK and SM MLCK but can be also expressed as an independent protein capable of stabilizing myofilaments in vitro (3,(21)(22)(23). The C-terminal half of the endothelial MLCK isoform (residues 923-1914) exhibits 99.8% homology to the human low molecular weight MLCK from hippocampus and substantial homology to published SM MLCK sequences from rabbit (94% homology), bovine (95% homology), and chicken (85% homology) (5, 6, 24, 25). However, the exact biological function of the 922-amino acid N-terminal portion, which is unique to the high

show abstract

Section: Identification Of P60mentioning

confidence: 73%

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Birukov

Csortos

Marzilli

et al. 2001

Journal of Biological Chemistry

148

140

View full text Add to dashboard Cite

show abstract

“…Src kinases preferentially phosphorylate peptide substrates containing SH3 ligands (41), but ACK1 showed no activity toward these peptides, although ACK1 is able to bind to the SH3-binding substrate ( Fig. 2A).…”

Section: Discussionmentioning

confidence: 99%

“…Finally, we included a substrate that possesses an SH3 domain-binding polyproline sequence. For Src family kinases, the addition of an SH3 domain ligand to a substrate increases its phosphorylation by activating down-regulated kinase and by decreasing substrate K m (41). The purified ACK1 kinase-SH3-CRIB construct clearly preferred the Abl substrate from this group of peptides ( Fig.…”

Section: Purification Of Ack1 and Characterization Of Substratementioning

confidence: 91%

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

“…Distributive phosphorylation of MAPKs is important for establishing, for example, the switch-like response observed in Xenopus oocytes in response to progesterone [45]. In the case of processive phosphorylation, Mayer et al first showed that Src homology 2 (SH2) domains promote hyperphosphorylation of substrates by tyrosine kinases [46], and subsequent model experiments confirmed that Src family kinases can phosphorylate substrates containing an SH2 domain ligand in a processive manner [47]. In this review, we discuss specific examples of proteins that undergo a processive mechanism of multisite phosphorylation.…”

Section: Multisite Phosphorylation By a Single Protein Kinasementioning

confidence: 99%

Processive phosphorylation: Mechanism and biological importance

Patwardhan

Miller

2007

Cellular Signalling

Self Cite

View full text Add to dashboard Cite

Recent proteomic data indicate that a majority of the phosphorylated proteins in a eucaryotic cell contain multiple sites of phosphorylation. In many signaling events, a single kinase phosphorylates multiple sites on a target protein. Processive phosphorylation occurs when a protein kinase binds once to a substrate and phosphorylates all of the available sites before dissociating. In this review, we discuss examples of processive phosphorylation by serine/threonine kinases and tyrosine kinases. We describe current experimental approaches for distinguishing processive from non-processive phosphorylation. Finally, we contrast the biological situations that are suited to regulation by processive and non-processive phosphorylation.

show abstract

A Peptide Model System for Processive Phosphorylation by Src Family Kinases

Cited by 51 publications

References 34 publications

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Biochemical Properties of the Cdc42-associated Tyrosine Kinase ACK1

Processive phosphorylation: Mechanism and biological importance

Contact Info

Product

Resources

About