Molecular basis for the substrate specificity of a serine threonine-specific protein kinase

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The active site substrate specificities of v-Abl and cSrc are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a "dual specificity" with respect to the ability to utilize D-and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.Protein phosphorylation plays a pivotal role in signaling pathways, which regulate such fundamental processes as cell growth, differentiation, and division (1, 2). Consequently, protein kinase inhibitors that can be precisely directed to specific members of the kinase family could prove to be of decided utility in understanding and controlling these signaling pathways. Much of the effort devoted to the design and construction of agents that are targeted to individual protein kinases has focused on the identification of characteristic substrate sequence specificities. Because most protein kinases utilize short peptides as substrates (3, 4), it may be possible to create inhibitors for specific protein kinases by employing peptides containing appropriate primary sequences. Unfortunately, this approach suffers from the limitation that many kinases exhibit broad overlapping sequence specificities with other family members. One way to overcome this obstacle is to employ additional recognition motifs in conjunction with the specific primary sequence to narrow the range of kinases that are impaired by inhibitory species.We have developed a facile method to rapidly assess the active site substrate specificity of protein kinases (5-11). Although protein kinases will generally phosphorylate only serine, threonine, and/or tyrosine residues in intact protein substrates, we have found that these enzymes will phosphorylate a diverse array of alcohol-containing non-amino ...

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Precision Substrate Targeting of Protein Kinases v-Abl and c-Src

Lee

Till

Lawrence

et al. 1995

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“…At first glance, this seems unlikely since both enzymes phosphorylate only serine and threonine residues in proteins. However, we have demonstrated that the active site specificities of several protein kinases are not merely limited to serine, threonine, and/or tyrosine (17)(18)(19)(20)(21)(22)(23). Indeed, PKA will phosphorylate a diverse array of alcohol-containing compounds, including phenols.…”

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confidence: 99%

Precision Substrate Targeting of Protein Kinases

Wood

Yan

Mendelow

et al. 1996

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The cAMP-dependent (PKA) and cGMP-dependent protein kinases (PKG) share a strong primary sequence homology within their respective active site regions. Not surprisingly, these enzymes also exhibit overlapping substrate specificities, a feature that often interferes with efforts to elucidate their distinct biological roles. In this report, we demonstrate that PKA and PKG exhibit dramatically different behavior with respect to the phosphorylation of ␣-substituted alcohols. Although PKA will phosphorylate only residues that contain an ␣-center configuration analogous to that found in L-serine, PKG utilizes residues that correspond to both L-and D-serine as substrates. The PKG/PKA selectivity of these substrates is the highest ever reported.Protein kinase cascades are the predominant component of signal transduction pathways (1). These pathways transmit extracellular signals from the plasma membrane to distant intracellular sites in the cytoplasm and nucleus. Estimates have placed the potential number of protein kinases encoded by the mammalian genome at greater than 1,000 (2). Consequently, defining the role of a particular protein kinase can be a daunting task given the size of this enzyme family and the plethora of pathways in which these phosphoryl transfer catalysts serve as participants. This task is rendered even more formidable by the general nature of the reaction catalyzed by these enzymes and by their nearly universal utilization of a common substrate, ATP. However, individual protein kinases presumably do exhibit unique properties in vivo, as exemplified by their ability to catalyze the phosphorylation of specific proteins. This precision is potentially regulated by several different parameters, including (i) the microenvironment to which the enzyme is confined within the cell (3), (ii) the secondary and/or tertiary structure that encompasses the site of phosphorylation on the target protein substrate, (iii) the primary sequence that envelops the phosphorylatable amino acid residue, and (iv) the active site substrate specificity of the protein kinase, which is generally limited to serine, threonine, and/or tyrosine residues (although exceptions are known).Can these four parameters be manipulated to design inhibitors that target specific protein kinases? Peptide-based substrates have been described for a number of protein kinases utilizing data from the specificity parameter (iii) described above (4). These substrate specificity studies have lead to the creation of nonphosphorylatable reversible inhibitors: peptides in which the alcohol-containing amino acid has been replaced by a residue that lacks a hydroxyl moiety (e.g. alanine for serine exchange). Unfortunately, the ultimate utility of this approach is limited by the fact that many protein kinases exhibit overlapping substrate specificities. A case in point is the cAMP-dependent protein kinase (5, 6) and its closely related counterpart, the cGMP-dependent protein kinase (6, 7). Both enzymes exhibit a special affinity for amino acid sequences that con...

“…In general, protein kinases will utilize aromatic or aliphatic alcohol-containing amino acids as substrates, but not both (although the dual specificity kinases are exceptions) (2). However, we have found that this restriction in substrate specificity vanishes in the context of non-amino acid residues (3)(4)(5)(6)(7)(8)(9)(10)(11). For example, the cAMP-dependent protein kinase (PKA) 1 catalyzes the phosphorylation of a wide assortment of synthetic aliphatic and aromatic alcohols (3,7).…”

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confidence: 99%

Is Protein Kinase Substrate Efficacy a Reliable Barometer for Successful Inhibitor Design?

Werner

Lee

Lawrence

1996

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We have addressed the question of whether protein kinase substrate efficacy is a reliable barometer for successful inhibitor design by assessing the dependence of kcat and kcat/Km for eight separate alcohol-bearing residues on solvent viscosity. We have found that the Km for three structurally distinct primary alcohol-containing peptides overestimates the affinity that these species exhibit for the cAMP-dependent protein kinase. In all three cases, the rate-determining step is product release, and substrate binding is best described as rapid equilibrium. In contrast, peptides containing the following phosphorylatable residues all provide Km values that are accurate assessments of substrate affinity for the protein kinase: a secondary alcohol, a simple phenol, and a primary alcohol with a relatively long side chain. In the latter three instances, the rate-determining step is phosphoryl transfer. Finally, two aromatic alcohol-containing residues that possess lipophilic side chains exhibit Michaelis constants that underestimate enzyme affinity. These results demonstrate that while it may be tempting to employ structural elements from the most efficient substrates (e.g. primary alcohols) for inhibitor design, less effective substrates may serve as a more accurate assessment of inhibitory success.