In-Gel Kinase Assay as a Method to Identify Kinase Substrates

Wooten, Marie W.

doi:10.1126/stke.2002.153.pl15

Cited by 29 publications

(14 citation statements)

References 9 publications

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“…S3A). To determine whether PEAK1 can phosphorylate an exogenous substrate, we performed an in-gel kinase assay by using myelin basic protein (MBP) as a generic substrate and γ-[ 32 P]ATP (36). Under these conditions PEAK1 was able to weakly, but consistently, phosphorylate MBP (Fig.…”

Section: Proteomic and Bioinformatic Analyses Of The Pseudopodial Pymentioning

confidence: 99%

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Wang¹,

Kelber²,

Cao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

108

190

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Regulation of the actin-myosin cytoskeleton plays a central role in cell migration and cancer progression. Here, we report the discovery of a cytoskeleton-associated kinase, pseudopodium-enriched atypical kinase 1 (PEAK1). PEAK1 is a 190-kDa nonreceptor tyrosine kinase that localizes to actin filaments and focal adhesions. PEAK1 undergoes Src-induced tyrosine phosphorylation, regulates the p130Cas-Crk-paxillin and Erk signaling pathways, and operates downstream of integrin and epidermal growth factor receptors (EGFR) to control cell spreading, migration, and proliferation. Perturbation of PEAK1 levels in cancer cells alters anchorage-independent growth and tumor progression in mice. Notably, primary and metastatic samples from colon cancer patients display amplified PEAK1 levels in 81% of the cases. Our findings indicate that PEAK1 is an important cytoskeletal regulatory kinase and possible target for anticancer therapy.

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Section: Proteomic and Bioinformatic Analyses Of The Pseudopodial Pymentioning

confidence: 99%

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Wang¹,

Kelber²,

Cao³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

108

190

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“…In gel kinase assay was performed as described previously (19,20) with minor modifications. Briefly, about 35-40 g of cytosolic protein from round spermatids was run on a 12% SDS-PAGE with the resolving gel containing 0.02% recombinant TP2.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of Rat Spermatidal Protein TP2 by Sperm-specific Protein Kinase A and Modulation of Its Transport into the Haploid Nucleus

Ullas

2003

Journal of Biological Chemistry

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Transition protein 2 (TP2), which is expressed during stages 12-15 of mammalian spermiogenesis, has been shown to undergo phosphorylation immediately after its synthesis. We reported earlier that TP2 is phosphorylated in vitro at threonine 101 and serine 109 by the salt extract of sonication-resistant (elongating and elongated) spermatid nuclei and the protein kinase phosphorylating TP2 was identified to be protein kinase A (PKA). We now report that the cytosol from haploid spermatids but not from premeiotic germ cells is able to phosphorylate recombinant TP2 in vitro at threonine 101 and serine 109. The kinase present in the haploid spermatid cytosol that phosphorylates TP2 has been identified to be the sperm-specific isoform of protein kinase A (Cs-PKA). Reverse transcription-PCR analysis indicated that Cs-PKA was present in the haploid spermatids and absent from premeiotic germ cells. The rat Cs-PKA transcript was amplified and sequenced using the isoform-specific primers. The sequence of rat Cs-PKA at the N terminus differs from mouse and human by one amino acid. Western blot analysis using specific anti-C␣1 antibodies revealed that C␣1-PKA is absent in haploid spermatid cytosol. We have also established an in vitro nuclear transport assay for the haploid round spermatids. Using this assay, we have found that the cytoplasmic factors and ATP are absolutely essential for translocation of TP2 into the nucleus. Phosphorylation was found to positively modulate the NLS dependent import of TP2 into the nucleus.The process of spermiogenesis in mammals, wherein the haploid round spermatids mature into highly condensed spermatozoa, can be broadly divided into three phases. In the first phase, encompassing stages 1-10, the round spermatids are transcriptionally active and contain nucleosomal chromatin. The second phase (stages 12-15) involves the replacement of nucleosomal histones by transition proteins TP1, TP2, and TP4.1 Finally, in the third phase, the transition proteins are replaced by protamines P1 and P2 during stages 16 -19 (1, 2). It is quite interesting to note that the transformation of nucleohistone type of chromatin into nucleoprotamine fiber is not complete, and ϳ10 -15% of sperm chromatin retains nucleosomal histones, probably in the context of nucleosomal structure (3-7). These unexpected observations have raised several interesting questions as to the nature of chromatin domains, which resist this replacement process, and the possible significance of the chromatin domains that retain nucleosomal structure during early embryonic development. Over the last several years, we have been studying the DNA binding and condensing properties of the spermatid protein TP2 and have described in detail its molecular anatomy with respect to the nature and identity of novel zinc finger domains, the nuclear localization signal, and also the role of phosphorylation in DNA condensation (8, 9). Based on our earlier observations, we have proposed a model describing the possible sequence of events from the time of TP2 synt...

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“…The In-gel kinase assays were done as described earlier [15] with minor modifications. Briefly, three sets of 3 μg purified CIITA and control cell extract (CE) were run on a 6% SDS-PAGE gel which was then cut into strips with one set of proteins each.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional coactivator CIITA, a functional homolog of TAF1, has kinase activity

Soe

Devaiah

Singer

2013

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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The Major Histocompatibility Complex (MHC) Class II Transactivator (CIITA) mediates activated immune responses and its deficiency results in the Type II Bare Lymphocyte Syndrome. CIITA is a transcriptional co-activator that regulates γ-interferon-activated transcription of MHC class I and class II genes. It is also a functional homolog of TAF1, a component of the general transcription factor complex TFIID. TAF1 and CIITA both possess intrinsic acetyltransferase (AT) activity that is required for transcription initiation. In response to induction by γ-Interferon, CIITA and it’s AT activity bypass the requirement for TAF1 AT activity. TAF1 also has kinase activity that is essential for its function. However, no similar activity has been identified for CIITA thus far. Here we report that CIITA, like TAF1, is a serine-threonine kinase. Its substrate specificity parallels, but does not duplicate, that of TAF1 in phosphorylating the TFIID component TAF7, the RAP74 subunit of the general transcription factor TFIIF and histone H2B. Like TAF1, CIITA autophosphorylates, affecting its interaction with TAF7. Additionally, CIITA phosphorylates histone H2B at Ser36, a target of TAF1 that is required for transcription during cell cycle progression and stress response. However, unlike TAF1, CIITA also phosphorylates all the other histones. The identification of this novel kinase activity of CIITA further clarifies its role as a functional homolog of TAF1 which may operate during stress and γ-IFN activated MHC gene transcription.

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In-Gel Kinase Assay as a Method to Identify Kinase Substrates

Cited by 29 publications

References 9 publications

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Pseudopodium-enriched atypical kinase 1 regulates the cytoskeleton and cancer progression

Phosphorylation of Rat Spermatidal Protein TP2 by Sperm-specific Protein Kinase A and Modulation of Its Transport into the Haploid Nucleus

Transcriptional coactivator CIITA, a functional homolog of TAF1, has kinase activity

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