(Received for publication, May 5, 1995; and in revised form, July 10, 1995)
From the
A human protein kinase (termed MST1) has been cloned and characterized. The MST1 catalytic domain is most homologous to Ste20 and other Ste20-like kinases (62-65% similar). MST1 is expressed ubiquitously, and the MST1 protein is present in all human cell lines examined. Biochemical characterization of MST1 catalytic activity demonstrates that it is a serine/threonine kinase, and that it can phosphorylate an exogenous substrate as well as itself in an in vitro kinase assay. Further characterization of the protein indicates MST1 activity increases approximately 3-4-fold upon treatment with PP2A, suggesting that MST1 is negatively regulated by phosphorylation. MST1 activity decreases approximately 2-fold upon treatment with epidermal growth factor; however, overexpression of MST1 does not affect extracellular signal-regulated kinase-1 and -2 activation. MST1 is unaffected by heat shock or high osmolarity, indicating that it is not involved in the stress-activated or high osmolarity glycerol signal transduction pathways. Thus MST1, although homologous to a member of a yeast MAPK cascade, is not involved in the regulation of a known mammalian MAPK pathway and potentially regulates a novel signaling cascade.
Regulation of cell growth and differentiation utilizes a complex
mechanism of signaling involving the catalytic activities of protein
kinases and phosphatases. The mechanisms used in signal transduction
are well conserved in all eukaryotes. In mammalian cells external
stimuli acting on both growth factor receptors and some G
protein-coupled receptors signal through a kinase cascade resulting in
the activation of members of the mitogen-activated protein kinase
(MAPK) ()family, ERK1 and
ERK2(1, 2, 3) . ERK1/2 phosphorylate a
variety of substrates including transcription factors and other
kinases(4, 5, 6) . Activation of ERK1/2
requires phosphorylation of both tyrosine and threonine residues which
is mediated by a dual specific kinase termed
MEK(7, 8) . MEK activation may represent a convergence
point for signaling since it is known that both Raf and MEKK
phosphorylate MEK(9, 10, 11) . Studies
involving pheromone signaling in both budding (Saccharomyces
cerevisiae) and fission (Schizosaccharomyces pombe) yeast
have revealed that similar signal transduction mechanisms operate in
these evolutionarily divergent organisms(12) . Additional
signal transduction cascades have since been identified in S.
cerevisiae and more recently in mammalian cells. These include
pathways responsible for cell wall biosynthesis, hyperosmotic sensing,
and spore formation in S. cerevisiae and those activated by
stress and high osmolarity in mammalian
cells(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
Structural homologies exist between the mammalian and yeast pathways at
the level of MAPK, MEK, and MEKK(11) . Complementation studies
in the pheromone response pathway with mammalian homologs indicate that
the proteins are also somewhat functionally
related(24, 25) .
In S. cerevisiae a
serine/threonine kinase termed Ste20 acts upstream of the MEKK, Ste11,
and downstream of the pheromone-linked G protein in the mating
pathway(26, 27) . The recent identification of rat and
human homologs to Ste20, termed p65, indicates that an
additional level of conservation exists among signal transduction
cascades from distantly related species (28) . (
)However, the identification of a Ste20 homolog in S.
cerevisiae, termed Sps1, which is involved in the spore formation
pathway, suggests that conservation at this level in the cascade may be
more complex(18) . Sps1, while similar to both Ste20 and
p65
throughout the catalytic domain, does not share any
significant homologies outside this domain.
We have cloned and
characterized a human protein kinase with a similar degree of homology
to Ste20, p65, Sps1, and a human protein kinase termed GC
kinase(29) . Because of its homology to Ste20 we have termed
this protein MST1 (Mammalian Sterile Twenty-like). Northern analysis indicates that MST1 is
ubiquitously expressed. Biochemical characterization of MST1 indicates
that it is a serine/threonine kinase and that it may be negatively
regulated by phosphorylation. MST1 does not function in the MAPK signal
transduction cascade nor is its activity increased in response to
growth factors, heat shock or high osmolarity suggesting that it is
involved in an as yet unidentified signal transduction pathway.
Figure 1: Nucleotide and predicted amino acid sequence of MST1. The proposed initiation codon is in bold. The region amplified using degenerate oligonucleotides is underlined. Amino acids (in one-letter code) and nucleotides are numbered at the left and right, respectively.
Figure 2:
A comparison of the MST1 catalytic domain
to Ste20, p65, Sps1, and GC kinase. A, the 11
subdomains conserved in protein kinases are indicated in roman
numerals above the sequences. The numbers at the right indicate amino acids. B, schematic shows the percent
amino acid similarity between MST1 and related kinases. The percentages
were obtained by comparing the catalytic domains of each deduced amino
acid sequence.
Figure 3: Expression pattern of MST1. A human multitissue blot containing poly(A) RNA (Clontech) was probed with nucleotides 178-481 encoding amino acids 55-154 of MST1 (A). B, the same blot probed with actin to check the integrity of the RNA.
Figure 4:
Characterization of the MST1 protein. A, Western blot analysis. Cell lysates from either AG876, 293,
HeLa, A431, Rat1A, PC12, NIH3T3, or COS cells (lanes
1-8, 15 µg/lane) MST1 immunoprecipitated from COS cells (lane 9) and immunoprecipitated Myc-epitope tagged MST1 (lanes 11) from transiently transfected COS cells (lane
10). Immunoblotting with the anti-MST1 antibody (m1-45) and
subsequent detection was performed as described under
``Experimental Procedures.'' B, in vitro kinase assays. Endogenous MST1 was immunoprecipitated from COS
cells with m1-45 (lane 1). As a negative control an
equal amount of protein was immunoprecipitated with preimmune serum (lane 2). Myc-MST1 was immunoprecipitated from transiently
transfected COS cells using the anti-myc antibody, 9E10 (lane 3). As a negative control COS cells were mock
transfected with the vector alone (lane 4).
Immunoprecipitations were incubated in kinase buffer containing
[-
P]ATP as described under
``Experimental Procedures.'' C, phosphoamino acid
analysis of in vitro phosphorylated MST1 (B, auto) and myelin basic protein (B, MBP).
Phosphoamino acids were resolved in one dimension using a pH 2.5
buffer(31) . Positions of unlabeled phosphoamino acids are
indicated below the autoradiograph.
Figure 5: MST1 does not activate ERK1/2. A-C, COS cells were transiently transfected with vector alone (lanes 1 and 2) or pCMV5-MMST1 (lanes 3 and 4), starved for 20 h, followed by treatment with EGF (400 ng/ml) for 10 min. Each lane contains 19 µg of lysate. A, lysates were separated in an SDS-polyacrylamide gel containing 0.25 mg/ml MBP followed by an in gel kinase assay as described under ``Experimental Procedures.'' B, Western blot using anti-MAPK antibodies. C, Western blot using anti-myc antibodies. The positions of ERK1/2 and full-length MST1 are indicated. D, kinetic analysis of endogenous MST1 following treatment with EGF for the indicated times. COS cells which had been starved for 20 h then treated with EGF were lysed and endogenous MST1 immunoprecipitated with the m1-45 antibody as described under ``Experimental Procedures.'' The upper panel is an in vitro kinase assay with MBP as the substrate, and the lower panel is a Western blot using the anti-MST1 antibody.
Western blot analysis of Myc-MST1 transfected COS cells with anti-Myc antibodies shows a protein migrating at 57 kDa consistent with the size of tagged MST1; however, a smaller protein (approximately 40 kDa) is also recognized that has significantly greater kinase activity than the p57 form (Fig. 5, A and C). Since the Myc-epitope is present on this faster migrating species, we conclude that it is a carboxyl-terminal truncation of MST1 and may indicate that a portion of the carboxyl terminus is inhibitory to kinase function. It is important to note that we do not see the p40 protein when MST1 is expressed from a weaker promoter (data not shown).
Figure 6: PP2A treatment stimulates MST1 kinase activity. COS cells were transiently transfected with vector alone, pJ3H-ERK1 or pJ3H-MST1, starved for 20 h, followed by treatment with EGF (400 ng/ml) for 10 min. After immunoprecipitation each sample with divided equally in two and treated with PP2A (0.5 unit) or mock treated with buffer alone as described under ``Experimental Procedures.'' A, PP2A treated immunoprecipitates were separated in an SDS-polyacrylamide gel containing 0.25 mg/ml MBP followed by an in gel kinase assay as described under ``Experimental Procedures.'' B, Western blot of indicated immunoprecipitates using anti-HA antibodies.
We have cloned and characterized a human protein kinase with considerable similarity to Ste20 and other Ste20-like kinases. Biochemical characterization of this kinase indicates that it is a prominent renaturable kinase in COS cells. We have tentatively named this kinase MST1 pending an assignment of a biological function. MST1 is expressed at approximately equal levels in all tissues. The MST1 protein is present in all human and monkey cell lines that we examined; however, it could not be detected in either rat cell line (Rat1A and PC12) and only at low levels in a mouse cell line (NIH3T3). We do not believe this is due to the inability of the MST1 polyclonal antiserum to recognize rodent MST1 since MST1 can be immunoprecipitated from NIH3T3 cells using the same antiserum (data not shown), rather MST1 is present at low levels in these lines.
An in vitro kinase assay using immunoprecipitated MST1 demonstrated that it can phosphorylate itself and an exogenous substrate, and phosphoamino acid analysis indicated that MST1 is a serine/threonine kinase. While MST1 kinase activity is readily detectable in an in vitro and in gel kinase assay, we do not believe MST1 is in its most active state for two reasons. First, treatment of MST1 immunoprecipitated from starved COS cells with PP2A results in an approximate 3-4-fold increase in kinase activity. We do not see any difference in MST1 activity throughout the cell cycle (data not shown); therefore, we do not believe that inhibition of activity is specific to quiescence. This result suggests that MST1 is held in a partially inactive state through serine/threonine phosphorylation. Second, a carboxyl-truncated form of MST1, arising apparently from proteolysis, is much more active than full-length MST1 (Fig. 5). This latter result indicates that the carboxyl terminus has an inhibitory role.
While the catalytic domains of MST1 and Ste20 are 64% similar, it is unable to complement a ste20 null allele in S. cerevisiae using a quantitative mating assay. Also, overexpression of MST1 does not activate ERK1/2 in COS cells. The inability of several growth factors, heat shock, or high osmolarity to increase MST1 activity indicates that MST1 is not involved in the MAPK or the HOG signal transduction cascades. The 2-3-fold decrease in MST1 activity following EGF stimulation, while modest, may indicate that MST1 is involved in a pathway which is affected by growth factors acting on tyrosine kinase receptors that is distinct from the MAPK pathway. However, because MST1 does not appear to be in its most active state, it is difficult to determine if a decrease in this low level of activity is biologically significant. The assignment of MST1 to a particular signal transduction cascade awaits identification of effectors of MST1 kinase activity and the target(s) of its catalytic activity.
At this time we do not
understand the significance of the homologies between MST1, Ste20, and
the other Ste20-like kinases. The kinase domains of all of these
proteins are quite conserved and represent what is becoming a growing
family of Ste20-like kinases. The high degree of similarity between
Ste20 and p65, including the conservation of the
Cdc42/Rac1 binding element, suggests that these proteins perform
similar functions and may represent a distinct subfamily, while MST1,
GC kinase, and SPS1 may be a part of another subfamily. Identification
of substrates for these Ste20-like kinases may help determine the
importance of these similarities.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18297[GenBank].