Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation.
Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is a...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3078918?pdf=render |
| _version_ | 1828813062061162496 |
|---|---|
| author | Arturo Sanz Daniela Ungureanu Tuija Pekkala Rob Ruijtenbeek Ivo P Touw Riet Hilhorst Olli Silvennoinen |
| author_facet | Arturo Sanz Daniela Ungureanu Tuija Pekkala Rob Ruijtenbeek Ivo P Touw Riet Hilhorst Olli Silvennoinen |
| author_sort | Arturo Sanz |
| collection | DOAJ |
| description | Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the K(m) for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or K(m) for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches. |
| first_indexed | 2024-12-12T09:53:55Z |
| format | Article |
| id | doaj.art-69ae644367c84bc2b99c88a21596b32e |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-12T09:53:55Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-69ae644367c84bc2b99c88a21596b32e2022-12-22T00:28:11ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0164e1852210.1371/journal.pone.0018522Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation.Arturo SanzDaniela UngureanuTuija PekkalaRob RuijtenbeekIvo P TouwRiet HilhorstOlli SilvennoinenJanus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the K(m) for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or K(m) for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.http://europepmc.org/articles/PMC3078918?pdf=render |
| spellingShingle | Arturo Sanz Daniela Ungureanu Tuija Pekkala Rob Ruijtenbeek Ivo P Touw Riet Hilhorst Olli Silvennoinen Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation. PLoS ONE |
| title | Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation. |
| title_full | Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation. |
| title_fullStr | Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation. |
| title_full_unstemmed | Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation. |
| title_short | Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation. |
| title_sort | analysis of jak2 catalytic function by peptide microarrays the role of the jh2 domain and v617f mutation |
| url | http://europepmc.org/articles/PMC3078918?pdf=render |
| work_keys_str_mv | AT arturosanz analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation AT danielaungureanu analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation AT tuijapekkala analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation AT robruijtenbeek analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation AT ivoptouw analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation AT riethilhorst analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation AT ollisilvennoinen analysisofjak2catalyticfunctionbypeptidemicroarraystheroleofthejh2domainandv617fmutation |